Circular dna molecule having a conditional origin of replication, process for their preparation and their use in gene therapy

ABSTRACT

A circular DNA molecule, useful for gene therapy, comprising at least one nucleic acid sequence of interest, characterised in that the region allowing the replication thereof has an origin of replication with a functionality in a host cell that requires the presence of at least one specific protein foreign to said host cell. A method for preparing same, cells incorporating said DNA molecules and uses thereof in gene therapy are also described.

This application is a continuation of application Ser. No. 10/268,948,filed Oct. 11, 2002, which is a continuation-in-part of application Ser.No. 09/043,193, filed Mar. 13, 1998 (now U.S. Pat. No. 6,977,174), whichis a 371 of application no. PCT/FR96/01414, filed Sep. 13, 1996, each ofwhich is incorporated by reference herein.

The present invention relates to a novel conditional replication DNAmolecule which can be used in gene therapy or for the production ofrecombinant proteins.

Gene therapy consists in correcting a deficiency or an anomaly byintroducing genetic information into the affected organ or cell. Thisinformation may be introduced either in vitro into a cell extracted fromthe organ and then reinjected into the body, or in vivo, directly intothe target tissue. As a molecule of high molecular weight and ofnegative charge, DNA has difficulty in spontaneously crossingphospholipid cell membranes. Various vectors are thus used in order toenable gene transfer to take place: viral vectors, on the one hand, andnatural or synthetic chemical and/or biochemical vectors, on the otherhand.

Viral vectors (retroviruses, adenoviruses, adeno-associated viruses,etc.) are very effective, in particular for crossing membranes, butpresent a certain number of risks such as pathogenicity, recombination,replication, immunogenicity, etc.

Chemical and/or biochemical vectors allow these risks to be avoided (forreviews, see Behr, 1993, Cotten and Wagner 1993). These are, forexample, cations (calcium phosphate, DEAE-dextran, etc.) which act byforming precipitates with DNA, which may be “phagocytosed” by the cells.They may also be liposomes in which the DNA is incorporated and whichfuse with the plasma membrane. Synthetic gene-transfer vectors aregenerally lipids or cationic polymers which complex the DNA and formwith it a particle bearing positive surface charges. As illustrations ofvectors of this type, mention may be made in particular ofdioctadecylamidoglycylspermine (DOGS, Transfectam™) orN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA,Lipofectin™).

However, the use of chemical and/or biochemical vectors or naked DNAimplies the possibility of producing large amounts of DNA ofpharmacological purity. The reason for this is that in gene therapytechniques, the medicinal product consists of the DNA itself and it isessential to be able to manufacture, in suitable amounts, DNAs havingproperties which are appropriate for therapeutic use in man.

In the case of non-viral vectorology, the vectors used are plasmids ofbacterial origin. The plasmids generally used in gene therapy carry (i)an origin of replication, (ii) a marker gene such as a gene forresistance to an antibiotic (kanamycin, ampicillin, etc.) and (iii) oneor more transgenes with sequences necessary for their expression(enhancer(s), promoter(s), polyadenylation sequences, etc.).

However, the technology currently available is not entirelysatisfactory.

On the one hand, the risk remains of dissemination in the body. Thus, abacterium which is present in the body can, at low frequency, receivethis plasmid. There is a greater likelihood of this taking place if itinvolves an in vivo gene therapy treatment in which the DNA may bedisseminated in the body of the patient and may come into contact withbacteria which infect this patient or bacteria of the commensal flora.If the bacterium receiving the plasmid is an enterobacterium, such as E.coli, this plasmid can be replicated. Such an event then leads todissemination of the therapeutic gene. Insofar as the therapeutic genesused in gene therapy treatments can code, for example, for a lymphokine,a growth factor, an anti-oncogene or a protein whose function isdefective in the host and which thus makes it possible to correct agenetic defect, the dissemination of some of these genes could haveunforeseeable and worrying effects (for example if a pathogenicbacterium acquired a human growth factor gene).

On the other hand, the plasmids generally used in non-viral gene therapyalso possess a marker for resistance to an antibiotic (ampicillin,kanamycin, etc.). The bacterium acquiring such a plasmid thus has anundeniable selective advantage since any antibiotic therapy, using anantibiotic from the same family as that which selects the plasmidresistance gene, will lead to selection of the plasmid in question. Inthis respect, ampicillin belongs to the α-lactams, which is the familyof antibiotics which is most frequently used worldwide. The use inbacteria of selection markers which are not antibiotic-resistance geneswould thus be particularly advantageous. This would avoid the selectionof bacteria which may have received a plasmid carrying such a marker.

It is thus particularly important to seek to limit the dissemination oftherapeutic genes and resistance genes as much as possible.

The subject of the present invention is specifically to propose novelDNA molecules which can be used in gene therapy or for the production ofrecombinant proteins in vitro and which replicate only in cells whichcan complement certain functions of these non-viral vectors.

The invention also relates to a particularly effective method forpreparing these DNA molecules.

The DNA molecules claimed have the advantage of removing the risksassociated with dissemination of the plasmid, such as (1) replicationand dissemination, which can lead to uncontrolled overexpression of thetherapeutic gene, (2) dissemination and expression of resistance genes.The genetic information contained in the DNA molecules according to theinvention effectively comprises the therapeutic gene(s) and the signalsfor regulating its (their) expression, a functional conditional originof replication which greatly limits the host cell spectrum of thisplasmid, a selection marker of reduced size which is preferablydifferent from a gene which imparts resistance to an antibiotic and,where appropriate, a DNA fragment which allows the resolution of plasmidmultimers. The probability of these molecules (and thus the geneticinformation which they contain) being transferred to a microorganism,and maintained stably, is very limited.

Lastly, the vectors according to the invention, also referred to asminiplasmids on account of their circular structure, their reduced sizeand their supercoiled form, have the following additional advantages: onaccount of their size which is reduced in comparison with theColE1-derived plasmids used conventionally, the DNA molecules accordingto the invention potentially have better in vivo bioavailability. Inparticular, they have improved capacities of cell penetration anddistribution. Thus, it is acknowledged that the diffusion coefficient intissues is inversely proportional to the molecular weight (Jain, 1987).Similarly, in the cell, high molecular weight molecules have poorerpermeability across the plasma membrane. In addition, in order for theplasmid to pass into the nucleus, which is essential for its expression,the high molecular weight is also a disadvantage, the nuclear poresimposing a size limit for diffusion into the nucleus (Landford et al.,1986). The reduction in size of the non-therapeutic parts of the DNAmolecule (origin of replication and selection gene in particular)according to the invention also makes it possible to decrease the sizeof the DNA molecules. The part which allows the replication andselection of this plasmid in the bacterium (1.1 kb) is decreased by afactor of 3, counting, for example, 3 kb for the origin of replicationand the resistance marker vector part. This decrease (i) in molecularweight and (ii) in negative charge imparts improved tissue, cellular andnuclear bioavailability and diffusion to the molecules of the invention.

More precisely, the present invention relates to a circular DNAmolecule, which is useful in gene therapy, this molecule comprising atleast one nucleic acid sequence of interest, characterized in that theregion which allows its replication comprises an origin of replicationwhose functionality in a host cell requires the presence of at least onespecific protein which is foreign to the said host cell.

This DNA molecule may be in single- or double-stranded form andadvantageously possesses a supercoiled form.

For the purposes of the present invention, the host cells used can be ofvarious origins. They can be eukaryotic or prokaryotic cells. Accordingto a preferred embodiment of the invention, they are prokaryotic cells.

The replication of bacterial plasmids conventionally requires thepresence of at least one protein, which is coded for by the host cell,of the RNA polymerase, Rnase, DNA polymerase, etc. type. For the reasonsalready explained above, it is not possible to overcome entirely, withthis type of replication, any possible risks of dissemination in thetreated organism. Advantageously, the functionality of the origin ofreplication of the DNA molecule according to the invention requires thepresence of a specific protein which is foreign to the host cell. Thesignificance of this characteristic is to reduce the host spectrum ofthe claimed plasmid to specific strains that express this initiatorprotein. The DNA molecule developed within the context of the presentinvention thus advantageously possesses a so-called conditional originof replication.

The conditional origin of replication used according to the presentinvention may originate from plasmids or bacteriophages which share thefollowing characteristics: they contain in their origin of replicationrepeat sequences, or iterons, and they code for at least onereplication-initiating protein (Rep) which is specific to them. By wayof example, mention may be made of the conditional replication systemsof the following plasmids and bacteriophages: specific initiator plasmidor bacteriophage protein RK2 (Stalker et al., 1981) TrfA R1 (Ryder etal., 1981) RepA pSC101 (Vocke and Bastia, 1983) RepA F (Murotsu et al.,1981) protein E Rts1 (Itoh et al., 1982, 1987) RepA RSF1010 (Miao etal., 1995) RepC P1 (Abeles et al., 1984) RepA P4 (Flensburg andCalendar, 1987) alpha protein lambda (Moore et al., 1981) protein O phi82 (Moore et al., 1981) protein O from phi 82 phi 80 protein O from phi80

According to a preferred embodiment of the invention, the origin ofreplication used in the DNA molecules claimed is derived from a naturalE. coli plasmid referred to as R6K.

The replication functions of R6K are grouped together in a 5.5 kbp DNAfragment (FIG. 1) comprising 3 origins of replication α, β, and γ (γ andα providing 90% of the replication) and an operon coding for the πreplication-initiator proteins and the protein Bis. The minimum amountof genetic information required to maintain this plasmid at itscharacteristic number of copies (15 copies per genome) is contained intwo elements: the 400 bp of ori γ and the gene pir, whose product is theπ initiator protein.

Ori γ may be divided into two functional parts: the core part and theactivator element (FIG. 1). The core part, which is essential forreplication, contains the iterons (7 direct repeats of 22 bp) to whichthe π protein represented in SEQ ID No. 1 becomes bound, and flankingsegments, which are targets of the host proteins (IHF, DnaA).

According to a preferred mode of the invention, the origin ofreplication of the vector claimed consists entirely or partially of thisγ origin of replication of the plasmid R6K and more preferably, entirelyor partially of SEQ ID No. 1 or one of its derivatives.

For the purposes of the present invention, the term derivative denotesany sequence which differs from the sequence considered on account ofdegeneracy of the genetic code, obtained by one or more modifications ofgenetic and/or chemical nature, as well as any sequence which hybridizeswith these sequences or fragments thereof and whose product possessesthe activity indicated with regard to the replication-initiator proteinπ. The term modification of the genetic and/or chemical nature may beunderstood to refer to any mutation, substitution, deletion, additionand/or modification of one or more residues. The term derivative alsocomprises the sequences homologous with the sequence considered, derivedfrom other cellular sources and in particular cells of human origin, orfrom other organisms, and possessing an activity of the same type. Suchhomologous sequences may be obtained by hybridization experiments. Thehybridizations may be performed starting with nucleic acid libraries,using the native sequence or a fragment thereof as probe, underconventional conditions of stringency (Maniatis et al., cf. Generaltechniques of molecular biology), or, preferably, under conditions ofhigh stringency.

The origin of replication described above, which has the advantage ofbeing of very limited size, is functional solely in the presence of aspecific initiator protein, protein Pi, produced by the gene pir (SEQ IDNo. 2). Since this protein can act in trans, it is possible tophysically dissociate the ori gamma from the pir gene, which may beintroduced into the genome of the cell which is chosen as the specifichost for these plasmids. Mutations in π may alter its inhibitoryfunctions (Inuzuka and Wada, 1985) and lead to an increase in the numberof copies of the R6K derivatives, up to more than 10 times the initialnumber of copies. These substitutions are all within a domain of 40amino acids, which therefore appears to be responsible for the controlby π of the number of plasmid copies (FIG. 2).

According to an advantageous embodiment of the present invention, the πprotein, expressed in the host cell, results from the expression of thegene represented in SEQ ID No. 2 or one of its derivatives as definedabove and more particularly of the gene pir 116 which comprises amutation when compared with the pir gene. This mutation corresponds tothe replacement of a proline by a leucine. In this context, the numberof copies of the R6K derivatives is about 250 copies per genome.

Besides a conditional origin of replication as defined above, the DNAmolecules claimed contain a region comprising one (or more) gene(s)which make it possible to ensure selection of the DNA molecule in thechosen host.

This may be a conventional marker of gene type which imparts resistanceto an antibiotic, such as kanamycin, ampicillin, chloramphenicol,streptomycin, spectinomycin, lividomycin or the like.

However, according to a preferred embodiment of the invention, thisregion is different from a gene which imparts resistance to anantibiotic. It may thus be a gene whose product is essential for theviability of the host envisaged, under defined culturing conditions. Itmay be, for example:

-   -   a gene coding for a suppressor tRNA, of natural or synthetic        origin. This is, more preferably, an amber codon tRNA (TAG)    -   a gene whose product is necessary for metabolism of the cell,        under certain culturing conditions, namely a gene involved in        the biosynthesis of a metabolite (amino acid, vitamin, etc.), or        a catabolism gene which makes it possible to assimilate a        substance present in the culture medium (specific nitrogen or        carbon source), etc.

According to a preferred mode of the invention, this region contains anexpression cassette of a gene coding for a suppressor tRNA for specificcodons. This latter may be chosen, in particular, from those coding forphenylalanine, cysteine, proline, alanine and histidine bases. It ismore particularly a suppressor tRNA for amber codons (TAG).

In this particular case, the system used to select, in the host cells,the DNA molecules which are the subject of the present inventionincludes two elements: 1) on the DNA molecule, a gene coding for asuppressor transfer RNA for the amber codon (TAG) which constitutes theselection marker, known as (sup) gene and 2) a specific host, one ofwhose genes, which is essential under certain culture conditions,contains an amber TAG codon. This cell may grow, under the cultureconditions for which the product of the gene containing the TAG codon isessential, only if the plasmid allowing the expression of sup is presentin the cell. The culture conditions thus constitute the pressure forselection of the DNA molecule. The sup genes used may be of naturalorigin (Glass et al., 1982) or may originate from a syntheticconstruction (Normanly et al., 1986, Kleina et al., 1990).

Such a system offers great flexibility insofar as, depending on the genecontaining an amber mutation, it is possible to determine variousselective media. In the bacterium Lactococcus lactis for example, theamber codon is located in a purine biosynthesis gene. This allows theselection of the plasmid carrying the gene coding for the suppressortRNA when the bacteria multiply in milk. Such a marker has the advantageof being very small and of containing no “foreign” sequences,originating from phages or transposons.

According to a particular embodiment of the invention, the DNA moleculealso comprises a DNA fragment, the target for site-specificrecombinases, which allows the resolution of plasmid multimers.

Thus, such a fragment, introduced on to a DNA molecule which is circularand whose origin of replication is, for example, ori gamma, allows theresolution of multimers of such a plasmid. Such multimers are observed,in particular, when the DNA molecule is prepared in a strain carrying amutated allele of pir, such as pir-116, which makes it possible toincrease the number of copies of the R6K derivatives.

This recombination may be achieved by means of various systems whichentail site-specific recombination between sequences. More preferably,the site-specific recombination of the invention is obtained by means ofspecific intramolecular recombination sequences which are capable ofrecombining with each other in the presence of specific proteins,generally referred to as recombinases. In this specific case, these arethe recombinases XerC and XerD. For this reason, the DNA moleculesaccording to the invention generally also comprise a sequence whichallows this site-specific recombination. The specific recombinationsystem present in the genetic constructions according to the invention(recombinases and specific recognition site) may be of differentorigins. In particular, the specific sequences and the recombinases usedmay belong to different structural classes, and in particular to thetransposon Tn3 resolvase family or to the bacteriophage lambda integrasefamily. Among the recombinases belonging to the transposon Tn3 family,mention may be made in particular of the resolvase of transposon Tn3 orof transposons Tn21 and Tn522 (Stark et al., 1992); the Gin invertase ofbacteriophage mu or alternatively plasmid resolvases, such as that ofthe par fragment of RP4 (Abert et al., Mol. Microbiol. 12 (1994) 131).Among the recombinases belonging to the bacteriophage ë integrasefamily, mention may be made in particular of the integrase of the phageslambda (Landy et al., Science 197 (1977) 1147), P22 and _(—)80 (Leong etal., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus influenzae(Hauser et al., J. Biol. Chem. 267 (1992) 6859), the Cre integrase ofphage P1, the integrase of plasmid pSAM2 (EP 350 341) or alternativelythe FLP recombinase of the 2í plasmid and the XerC and XerD recombinasesfrom E. coli.

Preferably, the DNA molecules which form the subject of the presentinvention contain the fragment cer from the natural E. coli plasmidColE1. The cer fragment used is a 382 bp HpaII fragment from ColE1 whichhas been shown to bring about, in cis, the resolution of plasmidmultimers (Summers et al., 1984; Leung et al., 1985). It is alsopossible to use a HpaII-TaqI fragment of smaller size (280 bp) or asmaller fragment (about 220 bp), contained in the HpaII fragment, whichfragments possess the same properties (Summers and Sherratt, 1988). Thisresolution takes place by way of a specific intramolecularrecombination, which involves four proteins encoded by the genome of E.coli: ArgR, PepA, XerC and XerD (Stirling et al., 1988, 1989; Colloms etal., 1990, Blakely et al., 1993).

In this respect, it is particularly advantageous to use all or part ofthe cer fragment of ColE1 or one of its derivatives as defined above.

According to an implementation variant, the DNA molecules of theinvention may also comprise a sequence capable of interactingspecifically with a ligand. Preferably, this is a sequence capable offorming, by hybridization, a triple helix with a specificoligonucleotide. This sequence thus makes it possible to purify themolecules of the invention by selective hybridization with acomplementary oligonucleotide immobilized on a support (see applicationWO 96/18744). The sequence can be positioned at any site in the DNAmolecule of the invention, provided that it does not affect thefunctionality of the gene of interest and of the origin of replication.

As a DNA molecule representative of the present invention, the plasmidpXL2774 and its derivatives may be claimed most particularly. For thepurposes of the invention, the term derivative is understood to refer toany construction derived from pXL2774 and containing one or more genesof interest other than the luciferase gene. Mention may also be made ofthe plasmids pXL3029 and 3030 containing an expression cassette of atherapeutic gene and a sequence capable of interacting specifically witha ligand.

The present invention also relates to the development of a process forthe construction of specific host cells, which are particularlyeffective for the production of these therapeutic DNA molecules.

Another subject of the present invention relates to a process for theproduction of a circular DNA molecule, characterized in that a host cellis cultured containing at least one DNA molecule as defined above and aprotein, which may or may not be expressed in situ, which conditions thefunctionality of the origin of replication of the said DNA molecule,which is specific and which is foreign to the said host cell, underconditions which allow the selection of host cells transformed by thesaid DNA molecules.

More preferably, the protein which conditions the functionality of theorigin of replication of the DNA molecule is expressed in situ from acorresponding gene. The gene coding for the replication-initiatingprotein may be carried by a subsidiary replicon, which is compatiblewith the derivatives of the conditional origin of replication used orwhich may be introduced into the genome of the host cell byrecombination, by means of a transposon, a bacteriophage or any othervector. In the particular case in which the gene expressing the proteinis placed on a subsidiary replicon, the latter also contains a promoterregion for functional transcription in the cell, as well as a regionwhich is located at the 3′ end and which specifies a transcriptiontermination signal. As regards the promoter region, this may be apromoter region which is naturally responsible for expressing the geneunder consideration when the latter is capable of functioning in thecell. It may also be a case of regions of different origin (responsiblefor expressing other proteins), or even of synthetic origin. Inparticular, it may be a case of promoter sequences for prokaryotic orbacteriophage genes. For example, it may be a case of promoter sequencesobtained from the cell genome.

As genes coding for the replication-initiating protein, use may be madeeither of wild-type genes or of mutated alleles which make it possibleto obtain an increased number of copies of the plasmids (or derivatives)specific for the initiator protein which conditions the functionality ofthe origin of replication used in the DNA molecule.

Such mutants have been described in particular for the plasmids R6K(Inuzuka and Wada, 1985; Greener et al., (1990), Rts1 (Terawaki andItoh, 1985, Terawaki et al., 1990; Zeng et al., 1990), F (Seelke et al.,1982; Helsberg et al., 1985; Kawasaki et al., 1991), RK2 (Durland etal., 1990; Haugan et al., 1992, 1995), pSC101 (Xia et al., 1991; Goebelet al., 1991; Fang et al., 1993).

In the particular case in which the DNA molecule used possesses anorigin of replication derived from the plasmid R6K, the initiatorprotein is a derivative of the π protein of this same plasmid. It isparticularly advantageous to express a mutated form of this proteinwhich is capable of increasing the number of initial copies appreciably.To do this, the gene incorporated into the host cell is preferablyrepresented by all or part of the sequence represented in SEQ ID No. 2or one of its derivatives and more preferably by the pir 116 gene. Theassociated mutation corresponds to the replacement of a proline by aleucine. According to a particular embodiment of the invention, this pir116 gene is directly incorporated into the host cell genome.

Advantageously, one of the genes of the specific host cell, which isessential under the culture conditions chosen, contains a specific codonwhich is recognizable by the selected suppressor tRNA in the DNAmolecule. According to a preferred mode of the invention, this is anamber TAG codon. In this particular case, the cell may grow, underculture conditions for which the product of the gene containing the TAGcodon is essential, only if the plasmid allowing the expression of supis present in the host cell. The culture conditions thus constitute thepressure for selection of the DNA molecule.

Preferably, the gene containing the amber codon is a gene involved inthe biosynthesis of an amino acid, arginine. This gene, argE, codes foran N-acetylornithinase (Meinnel et al., 1992) and in this case containsa TAG codon corresponding to a point mutation Gln-53 (CAG)->TAG; thepressure for selection of the plasmid carrying the sup gene is thenprovided by culturing in minimal M9 medium (Maniatis et al., 1989).However, this could also be, for example, a gene for biosynthesis of avitamin or a nucleic acid base, or alternatively a gene which allows aspecific nitrogen or carbon source to be used or any other gene whosefunctionality is essential for cellular viability under the chosenculture conditions.

The host cell is preferably chosen from E. coli strains and is morepreferably represented by the strain E. coli XAC-1.

According to a specific embodiment of the invention, the host cell usedin the claimed process is a cell of the E. coli strain XAC-1, containingthe pir 116 gene in its genome and transformed by the plasmid pXL2774 orone of its derivatives.

According to an advantageous variant of the invention, the host cellused in the process claimed is a prokaryotic cell in which the end A1gene or a homologous gene is inactivated. The endA gene codes forendonuclease I of E. coli. This periplasmic enzyme has a non-specificactivity of cleaving double-stranded DNA (Lehman, I. R., G. G. Roussosand E. A. Pratt (1962) J. Biol. Chem. 237: 819-828; Wright M. (1971) J.Bacteriol. 107: 87-94). A study carried out on various strains ofEscherichia coli (wild-type or endA) showed that the degradation ofplasmid DNA incubated in extracts of these bacterial strains existed inthe endA+ strains but not in the endA mutants. (Wnendt S. (1994)BioTechniques 17: 270-272). The quality of the plasmid DNA isolated fromendA+ strains or from endA mutants was studied by the company Promegausing their purification system (Shoenfeld, T., J. Mendez, D. Storts, E.Portman, B.†Patterson, J. Frederiksen and C. Smith. 1995. Effects ofbacterial strains carrying the endA1 genotype on DNA quality isolatedwith Wizard plasmid purification systems. Promega notes 53). Theirconclusion is as follows: the quality of the DNA prepared from endAmutants is, overall, better than that of DNA prepared in the endA+strains tested.

The quality of the plasmid DNA preparations is thus affected by anycontamination with this endonuclease (relatively long-term degradationof the DNA).

The deletion or mutation of the endA gene can be envisaged withoutdifficulty insofar as the mutants no longer having this endonucleaseactivity behave on the whole like wild-type bacteria (Dürwald, H. and H.Hoffmann-Berling (1968) J. Mol. Biol. 34: 331-346).

The endA 1 gene can be inactivated by mutation, total or partialdeletion, disruption, etc. Inactivation of the endA gene of the E. colistrain chosen to produce the pCOR plasmids can be achieved moreparticularly by transferring, by means of the P1 bacteriophage, theΔendA::Tc^(R) deletion described by Cherepanov and Wackernagel(Cherepanov, P. P. and W. Wackernagel. 1995. Gene disruption inEscherichia coli: Tc^(R) and Km^(R) cassettes with the option ofFlp-catalyzed excision of the antibiotic-resistance determinant. Gene158:9-14) or by exchanging the wild-type allele present in the genome ofthe bacterium of interest with a mutated or deleted allele of endA, byhomologous recombination. The use of this type of strain in the contextof the present invention makes it possible advantageously to improve thequality of the DNA produced.

The invention also relates to any recombinant cell containing a DNAmolecule as defined above. This may be a cell of various origins, ofeukaryotic, prokaryotic, etc. type.

According to another embodiment of the invention, the E. coli XAC-1 hostcell used in the process claimed is designated TEX1, and comprises atraD gene, or a homologous gene thereof, inactivated to abolish F′transfer. The traD is at the 5′ end of one of the tra operons andencodes a 81.7 kDa membrane protein that is directly involved in DNAtransfer and DNA metabolism (Frost et al., Microbiology Reviews, 1994,58: 162-210). traD mutants do not transfer DNA (Panicker et al., J.Bacteriol., 1985, 162:584-590). The episomal traD gene may beinactivated by mutation, total or partial deletion, or disruption usingmethods well known to those of skill in the art (See Example 9). Onemethod of inactivating this gene is described in Example 1, and theresulting E. coli XAC-1 pir116 endA⁻ traD⁻ strain so obtained isdesignated TEX1 (Soubrier et al., Gene Therapy, 1999, 6: 1482-1488).

According to one embodiment of the invention, the host cell used in theclaimed process is a cell of the E. coli strain XAC-1, containing thepir116 mutation combined with the pir42 mutation. The pir116 and pir42mutations affect different domains of the π protein. The pir116 mutationaffects the copy number control region, whereas the pir42 mutationaffects the putative leucine zipper motif, as displayed in FIG. 11. Thenucleotide and amino acid sequences of the pir gene containing thepir116 and pir42 mutations are set forth in FIG. 12 and SEQ ID NOs: 21and 22, respectively. The p42 mutation comprises a C to T transition atposition 124 from the N-terminal methionine (as referenced by the firstamino acid of the sequence), and thus results in substitution of theproline at position 42 by a leucine. The pir42 mutation was described byby Miron et al. (Proc Natl Acad Sci USA, 1994. 91(14): p. 6438-42; EMBOJ, 1992. 11(3): p. 1205-16), and was reported to increase the copynumber of an “ori gamma R6K-Km^(R)-pir42” plasmid by 2.5 fold ascompared to the same plasmid harboring the wild-type pr gene. Howeverthe p42 mutation was never used or described in combination with thepir116 mutation and while other mutations such as cop21 in the pir genecombined with the pir116 do not exhibit an increase of the plasmid copynumber, combination of the pir116 and pir42 mutations in a E. coli XAC-1endA⁻ traD⁻ strain surprisingly showed a significant increase of theplasmid copy number. Applicants have thus shown unexpected results ofthis combination in terms of copy number of the plasmids produced in E.coli host strains comprising the mutated pir116 and pir42 gene ascompared with strains harboring pir116 alone, or in a host cellcomprising the pir116 mutation combined with another mutation of the pirgene, such as the mutation cop21 (Inuzuka et al., FEBS Lett, 1988.228(1): p. 7-11). For example, E. coli TEX1pir42 (=XAC-1 endA⁻ traD⁻pir116 pir42) exhibited a 2-5 fold increase in the number of plasmid, ascompared to pir116 strain, or strains comprising combined pir116 andcop21 mutations (See Example 11).

According to another embodiment of the present invention, the host cellused in the process claimed is a prokaryotic host cell in which the recAgene or a homologous gene has been inactivated. Preferably, the hostcell according to the present invention is E. coli strain XAC-1comprising mutations pir116, pir42, endA, traD, recA^(−.) Such a strainis designated TEX2pir42. recA may be inactivated by methods well knownto those in the art. recA encodes a major recombination protein andmutations in this gene reduce the frequency of recombination-mediatedalteration in plasmids and intramolecular recombination that could leadto the multimerization of plasmids. As described in Example 12, adeleted recA gene containing 3 translation stop codons (one in eachframe) at its 5′ end may be obtained by PCR. The resulting inactivatedgene was then introduced by gene replacement into TEX1 genome (Example12.1).

These cells are obtained by any technique known to those skilled in theart which allows the introduction of the said plasmid into a given cell.Such a technique may be, in particular, transformation, electroporation,conjugation, fusion of protoplasts or any other technique known to thoseskilled in the art.

Strain TEX1 was deposited under the terms of the Budapest Treaty withthe Collection Nationale De Cultures De Micro-organismes (CNCM),Institut Pasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15, France, onDec. 10, 2004, under accession no. CNCM I-3343.

Strain TEX2pir42 was deposited under the terms of the Budapest Treatywith the Collection Nationale De Cultures De Micro-organismes (CNCM),Institut Pasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15, France, onOct. 10, 2003, under accession no. CNCM I-3109.

Strain TEX2 was deposited under the terms of the Budapest Treaty withthe Collection National De Cultures De Micro-organismes (CNCM), InstitutPasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15, France, on Dec. 10,2004, under accession no. CNCM I-3344.

Strain XAC1pir116 was deposited under the terms of the Budapest Treatywith the Collection Nationale De Cultures De Micro-organismes (CNCM),Institut Pasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15, France, onOct. 10, 2003, under accession no. CNCM I-3108.

The DNA molecules according to the invention may be used in anyapplication of vaccination or of gene and cell therapy for transferringa gene to a given cell, tissue or organism, or for the production ofrecombinant proteins in vitro.

In particular, they may be used for direct in vivo administration or forthe modification of cells in vitro or ex vivo, for the purpose ofimplanting them into a patient.

In this respect, another subject of the present invention relates to anypharmaceutical composition comprising at least one DNA molecule asdefined above. This molecule may or may not be associated therein with achemical and/or biochemical transfection vector. This may in particularinvolve cations (calcium phosphate, DEAE-dextran, etc.) or liposomes.The associated synthetic vectors may be cationic polymers or lipids.Examples of such vectors which may be mentioned are DOGS (Transfectam™)or DOTMA (lipofectin™).

The pharmaceutical compositions according to the invention may beformulated for the purpose of topical, oral, parenteral, intranasal,intravenous, intramuscular, subcutaneous, intraocular, or transdermaladministrations. The claimed plasmid is preferably used in an injectableform or in application. It may be mixed with any vehicle which ispharmaceutically acceptable for an injectable formulation, in particularfor a direct injection to the site to be treated. This may involve, inparticular, sterile, isotonic solutions or dry compositions, inparticular freeze-dried compositions, which, by addition, depending onthe case, of sterilized water or of physiological saline, allowinjectable solutions to be made up. This may in particular involve Trisor PBS buffers diluted in glucose or in sodium chloride. A directinjection into the affected region of the patient is advantageous sinceit allows the therapeutic effect to be concentrated at the level of theaffected tissues. The doses used may be adapted as a function of variousparameters, and in particular as a function of the gene, the vector, themode of administration used, the pathology concerned or the desiredduration of the treatment.

The DNA molecules of the invention may contain one or more genes ofinterest, that is to say one or more nucleic acids (synthetic orsemi-synthetic DNA, gDNA, cDNA, etc.) whose transcription and, possibly,whose translation in the target cell generate products of therapeutic,vaccinal, agronomic or veterinary interest.

Among the genes of therapeutic interest which may be mentioned moreparticularly are genes coding for enzymes, blood derivatives, hormonesand lymphokines: interleukins, interferons, TNF, etc. (FR 92/03120),growth factors, neurotransmitters or their precursors or syntheticenzymes, and trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF,NT3, NT5, VEGF-B, VEGF-C etc.; apolipoproteins: ApoAI, ApoAIV, ApoE,etc. (FR 93/05125), dystrophin or a minidystrophin (FR 91/11947),tumour-suppressing genes: p53, Rb, Rap1A, DCC, k-rev, etc. (FR93/04745), genes coding for factors involved in coagulation: factorsVII, VIII, IX, etc., suicide genes: thymidine kinase, cytosinedeaminase, etc.; or alternatively all or part of a natural or artificialimmunoglobulin (Fab, ScFv, etc.), an RNA ligand (WO 91/19813), etc. Thetherapeutic gene may also be an antisense sequence or gene, whoseexpression in the target cell makes it possible to control theexpression of genes or the transcription of cellular mRNAs. Suchsequences may, for example, be transcribed, in the target cell, intoRNAs which are complementary to cellular mRNAs and thus block theirtranslation into protein, according to the technique described in patentEP 140,308.

The gene of interest may also be a vaccinating gene, that is to say agene coding for an antigenic peptide, capable of generating an immuneresponse in man or animals, for the purpose of producing vaccines. Theseantigenic peptides may in particular be specific antigenic peptides ofEpstein-Barr virus, HIV virus, hepatitis B virus (EP 185,573), orpseudorabies virus, or alternatively specific antigenic peptides oftumours (EP 259,212).

Generally, in the DNA molecules of the invention, the gene oftherapeutic, vaccinal, agronomic or veterinary interest also contains apromoter region for functional transcription in the target organism orcell, as well as a region located at the 3′ end which specifies atranscription termination signal and a polyadenylation site. As regardsthe promoter region, it may be a promoter region naturally responsiblefor expression of the gene under consideration when this region iscapable of functioning in the cell or the organism concerned. Thepromoter regions may also be regions of different origin (responsiblefor the expression of other proteins) or even of synthetic origin. Inparticular, they may be promoter sequences from eukaryotic or viralgenes. For example, they may be promoter sequences obtained from thegenome of the target cell. Among the eukaryotic promoters which may beused are any promoter or derived sequence which stimulates or suppressesthe transcription of a gene in a specific or non-specific, inducible ornon-inducible, strong or weak manner. The eukaryotic promoters may inparticular be ubiquitous promoters (promoters of the genes for HPRT,PGK, α-actin, tubulin, etc.), intermediate filament promoters (promotersof the genes for GFAP, desmin, vimentin, neurofilaments, keratin, etc.),therapeutic gene promoters (for example the promoters of the genes forMDR, CFTR, factor VIII, ApoAI, etc.) tissue-specific promoters(promoters of the genes for pyruvate kinase, villin, intestinal fattyacid-binding protein, à-actin of smooth muscle, etc.) or alternativelypromoters which respond to a stimulus (steroid hormone receptor,retinoic acid receptor, etc.). Similarly, they may be promoter sequencesobtained from the genome of a virus, such as, for example, the promotersof the adenovirus E1A and MLP genes, the CMV early promoter oralternatively the LTR promoter of RSV, etc. In addition, these promoterregions may be modified by addition of activating or regulatorysequences or sequences which allow tissue-specific expression orexpression which is predominantly tissue-specific.

Moreover, the gene of interest may also contain a signal sequence whichdirects the synthesized product into the secretory pathways of thetarget cell. This signal sequence may be the natural signal sequence ofthe synthesized product, but it may also be any other functional signalsequence or an artificial signal sequence.

Depending on the gene of interest, the DNA molecules of the inventionmay be used for the treatment or prevention of several pathologies,including genetic diseases (dystrophy, cystic fibrosis, etc.),neurodegenerative diseases (Alzheimer's disease, Parkinson's disease,ALS, etc.), cancers, pathologies associated with coagulation disordersor with dyslipoproteinaemias, pathologies associated with viralinfections (hepatitis, AIDS, etc.), or in the agronomic and veterinaryfields, etc.

Moreover, the present invention also relates to the use of conditionalreplication DNA molecules for the production of recombinant proteins.Bacteria can be used to produce proteins of various origins, eukaryoticor prokaryotic. Among the bacteria, E. coli constitutes the organism ofchoice for expressing heterologous genes on account of its ease ofmanipulation, the large number of expression systems available and thelarge amounts of proteins which can be obtained. It is understood thatthe system of the invention can be used in other organisms, the tropismbeing determined by the nature of the origin of replication, asindicated above. For this use, the nucleic acid sequence of interestcomprises a coding region under the control of expression signals thatare appropriate for the host chosen, in particular a prokaryotic host.These may be, for example, Plac, Ptrp, PT7, Ptrc, Ptac, PL or PRpromoters, the Shine-Dalgarno sequence, etc. (this set constitutes theexpression cassette). The nucleic acid sequence of interest can be anysequence coding for a protein which is of value in the fields ofpharmacy, agri-foods, chemistry or agrochemistry. This may be astructural gene, a complementary DNA sequence, a synthetic orsemi-synthetic sequence, etc.

The expression cassette can be introduced onto the conditionalreplication vector which is the subject of the invention, thusconstituting a conditional replication vector which allows theexpression of proteins of interest in E. coli. This vector has severaladvantages: no use of antibiotic to select it in the bacterium (reducedcost, no need for a study regarding the presence of antibiotic or ofpotentially toxic derived products in the finished product), virtuallyno probability of dissemination of the plasmid in nature (origin ofconditional replication), possible fermentation in entirely definedmedium. The examples given show the advantageous properties of theseconditional vectors for the production of recombinant proteins.

The present invention will be described more fully with the aid of theexamples which follow, which should be considered as non-limitingillustrations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Functional organization of the region of R6K involved inreplication.

FIG. 2: Organization of the functional domains of the π protein of theplasmid R6k.

FIG. 3: Representation of the protocol for introducting the pir geneinto the genome of E. coli XAC1.

FIG. 4: Construction scheme for vectors pXL2666, 2730 and 2754.

FIG. 5: Construction of pXL2774.

FIG. 6: Growth and production kinetics in a 2L fermenter.

FIG. 7: Growth and production kinetics in an 800L fermenter.

FIG. 8: Construction of pXL3056.

FIG. 9: Visualization of the aFGF protein produced by E. coliXAC-1pir-116 (pXL3056+PT7pol23) after induction. The denatured totalcell extracts are deposited on 12.5%-SDS polyacrylamide gel. M:molecular mass marker (Biorad, Low range). Each band is identified by anarrow and a figure which indicates its mass in kdaltons. 1: XAC-1pir-116(pXL3056+pUC4K) not induced; 2: XAC-1pir-116 (pXL3056+pUC4K) induced at42° C.; 3: XAC-1pir-116 (pXL3056+PT7pol23) clone 1, not induced; 4:XAC-1pir-116 (pXL3056+PT7pol23) clone 1, induced at 42° C.; 5:XAC-1pir-116 (pXL3056+PT7pol23) clone 2, not induced; 6: XAC-1pir-116(pXL3056+PT7pol23) clone 2, induced at 42° C.; t1: 1 μg of purifiedaFGF; t4: 4 μg of purified aFGF.

FIG. 10: Construction scheme for vectors pXL3029 and pXL3030.

FIG. 11: Schematic representation of the functional domains of R6K πinitiator proteins.

FIG. 12: Nucleotide and amino acid sequences of the pir gene comprisingthe pir116 and pir42 mutations.

FIG. 13: Construction of pir116-pir42 suicide vector for homologousrecombination.

FIG. 14: Schematic representation of the PCR products obtained whenamplifying the region uidA::pir116+/−pir42.

FIG. 15: Agarose gel electrophoresis showing the topology of pCORplasmid pXL3179 produced in TEX1 or TEX1pir42.

FIG. 16: Schematic representation of the pXL3749 suicide plasmidcarrying pir16-cop21 gene.

FIG. 17: Agarose gel electrophoresis showing the plasmid copy number ofpXL2979 when produced in E. coli host cell TEX1cop21 (lines 1-4), in E.coli host cell XAC1pir (lines 5-8), in E. coli TEX1 (lines 9-12).

FIG. 18: Representation of the cloning strategy for the construction ofthe recA-suicide vector.

FIG. 19: Schematic representation of the PCR products obtained whenamplifying regions of E. coli TEX2 strain.

FIG. 20: Agarose gel electrophoresis showing the topology of pCORpXL3179 produced in E. coli TEX2pir42 (line B), in E. coli TEX1pir42(line C), in E. coli TEX1 (line D).

FIG. 21: Analysis of plasmid pXL3179 produced by fermentation in E. coliTEX2pir42.

I—MATERIALS AND METHODS

-   A) Materials    -   1) Culture Media

Complete LB, 2XTY and SOC media and minimal M9 medium (Maniatis et al.,1989) were used. Agar media were obtained by addition of 15 g of Difcoagar. Furthermore, if necessary, these media were supplemented withantibiotics (ampicillin or kanamycin) at respective concentrations of100 mg/l and 50 mg/l. The chromogenic substrates X-Gal and X-Gluc wereused at a concentration of 40 mg/l.

-   -   2) E. coli Strains, Plasmids and Bacteriophages

The E. coli strains, plasmids and bacteriophages used are respectivelyidentified in the examples below.

-   B) Methods    -   1) Manipulation of the DNA

The isolation of bacterial DNA (plasmid and genomic) and phage DNA(replicative form of M13), digestion with restriction endonucleases,ligation of the DNA fragments, agarose gel electrophoresis (in TBEbuffer) and other standard techniques were carried out according to themanufacturers' recommendations, for the use of enzymes, or in accordancewith the procedures described in “Molecular Cloning: a LaboratoryManual” (Maniatis et al., 1989).

The DNA size markers used during the electrophoreses are as follows: 1kb ladder (BRL) for the linear fragments and the supercoiled DNA marker(Stratagene) for the undigested plasmids.

Sequencing was carried out according to the Sanger technique (Sanger etal., 1977) adapted to the automated method using fluorescentdideoxynucleotides and Taq DNA polymerase (PRISM Ready ReactionDyeDideoxy Terminator Cycle Sequencing Kit, Applied Biosystems).

The oligodeoxynucleotides used (designated by “seq+no.”, see below) weresynthesized on the “Applied Biosystems 394 DNA/RNA Synthesizer” by thephosphoramidite method, using α-cyanoethyl protecting groups (Sinha etal., 1984). After synthesis, the protecting groups are removed bytreatment with ammonia. Two precipitations with butanol allow theoligonucleotide to be purified and concentrated (Sawadogo et al., 1991).Sequences of the oligonucleotides used for the PCR amplification: SEQ IDNo. 3 5′-GACCAGTATTATTATCTTAATGAG-3′ SEQ ID No. 45′-GTATTTAATGAAACCGTACCTCCC-3′ SEQ ID No. 55′-CTCTTTTAATTGTCGATAAGCAAG-3′ SEQ ID No. 65′-GCGACGTCACCGAGGCTGTAGCCG-3′

The PCR reactions (Saiki et al., 1985) were performed under thefollowing conditions, in a total volume of 100 μl. The reaction mixturecomprises 150 ng of genomic DNA from the strain to be studied, 1 μg ofeach of the two oligonucleotide primers (24-mer), 10 μl of 10×PCRbuffer, the composition of which is as follows “500 mM KCl, 0.1%gelatin, 20 mM MgCl₂, 100 mM Tris-HCl pH 7.5”, and 2.5 units of Taq DNApolymerase (Amplitaq Perkin-Elmer). The PCR conditions, on thePerkin-Elmer Cetus DNA Thermal Cycler machine are as follows: 2 min at91° C., 30 successive cycles of denaturation (1 min at 91° C.),hybridization (2 min at 42° C.) and elongation (3 min at 72° C.), andfinally 5 min at 72° C. The products thus obtained, which are or are notdigested with a restriction enzyme, are analysed by agarose gelelectrophoresis.

Analysis of the various plasmid species by DNA topoisomerases wasperformed according to the following procedure: the enzymes, purified inthe laboratory, are incubated for 1 hour at 37° C. The reaction mixtures(total volume: 40 ìl) have the following composition: 150 ng of plasmid,300 ng of DNA topoisomerase I or 150 ng of E. coli DNA gyrase, or 160 ngof S. aureus DNA topoisomerase IV and 20 μl of buffer specific for eachenzyme. The composition of these buffers is indicated below:

for DNA topoisomerase I:

50 mM Tris-HCl pH 7.7, 40 mM KCl, 1 mM DTT, 100 μg/ml BSA, 3 mM MgCl₂, 1mM EDTA;

for DNA topoisomerase IV:

60 mM Tris-HCl pH 7.7, 6 mM MgCl₂, 10 mM DTT, 100 μ/ml BSA, 1.5 mM ATP,350 mM potassium glutamate;

for DNA gyrase:

50 mM Tris-HCl pH 7.7, 5 mM MgCl₂, 1.5 mM ATP, 5 mM DTT, 100 μg/ml BSA,20 mM KCl.

-   -   2) Transformation of E. coli

This was performed routinely according to the TSB (Transformation andStorage Buffer) method described by Chung and Miller (1988). For astrain such as TG1 (Gibson et al., 1984), the transformation efficiencyobtained is about 10⁵-10⁶ transformants per μg of pUC4K (Vieira andMessing; 1982). When a higher transformation efficiency was necessary,the bacteria were transformed by electroporation according to theprocedure recommended by the electroporator manufacturer (Biorad). Thismethod makes it possible to achieve efficiencies of from 10⁸ to 10¹⁰transformants per μg of pUC4K.

-   -   3) Cellular Transfection Mediated by a Cationic Lipofectant

The cells used are NIH 3T3 mouse fibroblasts seeded the day before into24-well plates, at a density of 50,000 cells per well. The culturemedium used is DMEM medium, containing 4.5 g/l of glucose andsupplemented with 10% foetal calf serum and 1% of solutions of 200 mMglutamine and antibiotics (5.10³ μ/ml streptomycin, 5.10³ μg/mlpenicillin) (Gibco). The plasmid DNA (1 μg in 25 μl of 9% NaCl) ismixed, on a volume-for-volume basis, with a suspension of lipofectant.Four “lipofectant charges/DNA charges” ratios are tested: 0, 3, 6 and 9.These ratios are calculated by considering that 1 μg of plasmid DNAcarries 3.1 nmol of negative charges and that the lipofectant contains 3positive charges per molecule. After a contact time of 10 minutes toallow formation of the DNA/lipid complex, 50 μl of DNA-lipofectantmixture are introduced onto the cells in serum-free culture medium (500μl). The cells were prerinsed twice with this same medium. Inhibition oftransfection by the serum is thus avoided. After incubation (2 hours at37° C. in the CO₂ incubator), 10% foetal calf serum is added to themedium. The cells are then reincubated for 24 hours.

-   -   4) Measurement of the Luciferase Activity of Eukaryotic Cells

This is carried out 24 hours after the transfection. Luciferasecatalyses the oxidation of luciferin in the presence of ATP, Mg²⁺ andO₂, with concomitant production of a photon. The total amount of lightemitted, measured by a luminometer, is proportional to the luciferaseactivity of the sample. The reagents used are supplied by Promega(luciferase assay system) and used according to the recommendedprocedure. After lysis of the cells, the insoluble fraction from eachextract is eliminated by centrifugation. The assay is carried out on 5μl of supernatant, which may or may not be diluted in the cell lysisbuffer.

-   -   5) Measurement of the Protein Concentration in the Cell Extracts

This is carried out according to the BCA method (Pierce) usingbicinchoninic acid (Wiechelman et al., 1988). The standard BSA range isprepared in the lysis buffer (cf. III-B-4). The samples to be assayedand those of the range are pretreated, on a volume-for-volume basis,with 0.1 M iodoacetamide/0.1 M Tris buffer, pH 8.2, for 1 hour at 37° C.This treatment makes it possible to prevent interference, during theassay, of the reducing agent (DTT) present in the lysis buffer. Theassay result is read at 562 nm.

EXAMPLE 1 Construction of XAC-1 pir and pir-116 Host Strains byHomologous Recombination

The strain used is the E. coli strain XAC-1 (Normanly et al., 1980). TheargE gene of this strain advantageously includes a mutation ofglutamine-53 (CAG) into the amber codon (TAG) (Meinnel et al., 1992).The argE gene belongs to the argECBH divergent operon and codes for anarginine biosynthesis enzyme, N-acetylornithinase. XAC-1 cannottherefore synthesize arginine and, consequently, grow in minimal medium.This auxotrophy will be relieved if the strain harbours a plasmid whichallows the expression of a suppressor tRNA. It will thus be possible, byculturing in minimal medium, to select bacteria which carry such aplasmid. In order to allow the replication therein of plasmids derivedfrom R6K, it was necessary to introduce, by homologous recombination,the pir gene into the genome of XAC-1. The pir gene (wild-type ormutated) is introduced at the uidA locus by exchange between thewild-type uidA gene and a copy interrupted by the pir (or pir-116) gene.The uidA gene codes for β-glucuronidase, the enzyme for hydrolysis ofβ-glucuronides. This gene may be inactivated without any problem sinceit is not essential for growth in standard synthetic media, in whichβ-glucuronides are not used. Furthermore, the β-glucuronidase activitycan be monitored by means of a chromogenic substrate, X-Gluc, whosehydrolysis releases a blue pigment.

-   -   1) Construction of a Suicide Vector Carrying the Cassette        “Km^(R)-uidA::pir (or pir-116)

We used a strategy involving a single bacterial host and minimizing themodifications to the genome of the strain of interest. The phage M13mp10(Messing et Vieira; 1982) was used as suicide vector (Blum et al.,1989). An amber mutation in the gene II, which is essential forreplication, reduces the host spectrum of this M13 to the strains, suchas TG1 (supE), which produce an amber suppressor tRNA; it will thereforenot be able to replicate in E. coli sup+ strains, such as XAC-1.

The 3.8 kb BamHI cassettes, containing the kanamycin-resistance gene ofTn5 and _uidA::pir or pir-116, were respectively purified from M13wm34and 33 (Metcalf et al., 1994). They were cloned into M13mp10 linearizedwith BamHI. The recombinant clones were selected by plating on LB+Kmagar medium, after electroporating the ligation mixtures into TG1. Theconformity of the clones obtained was shown by analysing the restrictionprofile and by sequencing the region corresponding to the pir-116mutation.

-   -   2) Introduction of the pir or pir-116 Genes into the Genome        of E. coli XAC-1 by Homologous Recombination

The strategy adopted and the various events involved are presented inFIG. 3.

-   -   -   a) First Recombination Event

The XAC-1 strain was transformed by electroporation with 10, 100 or 2000ng of each RF (mp10-_uidA::pir or pir-116). One-third of each expressionmixture was plated out on LB plates containing kanamycin and incubatedovernight at 37° C. The mp10-_uidA::pir or pir-116 phages cannotreplicate in the strain XAC-1 (sup+). The Km^(R) marker can thereforeonly be maintained by integration into the genome of the bacterium via ahomologous recombination with the wild-type copy of the gene uidA. Theresults of the electroporations of XAC-1 are presented in Table 1. Thetransformation efficiency obtained was 4.10⁹ transformants per μg ofpUC4K. TABLE 1 Number of colonies obtained with the amounts of DNAtransformed CONSTRUCT 10 ng 100 ng 2000 ng M13mp10-_uidA::pir 1 41 146M13mp10-_uidA::pir-116 0 16 124

Under the test conditions, the number of integrants increases in anon-linear manner with the amount of DNA. Given the transformationefficiency and the size of the RFs (11.7 kbp), it is possible to have anapproximate idea of the level of recombination. By considering the pointat 100 ng, a recombination frequency of about 10⁻⁶ is obtained.

-   -   -   b) Second Recombination Event

The second recombination event will then be selected by the resistanceof the strains to deoxycholate (Doc^(R)).

To do this, 5 integrants of each construct were cultured in 2XTY mediumsupplemented with 0.2% sodium deoxycholate. Two distinct populationsappeared. Certain clones give quite visible cloudiness after about 8hours at 37° C. (two clones for the pir construction and three for thepir-116 construction). The other clones gave a dense culture only afterone night at 37° C. They were virtually all Km^(S), as expected. Foreach of the electroporants studied, 50 Km^(S) descendants were streakedonto LB medium supplemented with X-Gluc. After 48 hours at 37° C., theUidA⁺ clones were pale blue whereas those which had undergone an allelereplacement (case No. 1, FIG. 3) remained white on this medium (UidA⁻).Table 2 summarizes the phenotype analysis of the double recombinantsobtained.

From 18 to 30% of the double recombinants underwent an allelereplacement. TABLE 2 Number of Km^(S) Percentage of UidA⁻ Strain amongthe Doc^(R) among the Km^(S) XAC-1 pir-2 50/50 18 XAC-1 pir-3 50/50 24XAC-1 pir-4 50/50 34 XAC-1 pir-116-1 50/50 32 XAC pir-116-4 35/50 30

-   -   3) Checking the Pir+ Character Nature of the Strains Obtained by        Recombination

In order to ensure the Pir+ character of the strains obtained by doublerecombination, we transformed three clones of each construct with pBW30(Metcalf et al., 1994). The fact that transformants were obtained forall the test strains made it possible to show the functionality of thepir and pir-116 genes which were integrated into the genome of XAC-1.Under the same conditions, no transformant is obtained with the parentalstrain XAC-1. We continued to study 2 XAC-1pir clones (B and C) and 2XAC-1pir-116 clones (E and D).

-   -   4) Checking, by PCR Amplification, of the Strains Obtained by        Recombination

In order to confirm the allele replacement, we checked the genomicregions on either side of the uidA locus by PCR amplification. Each pairof oligonucleotides consists of an oligonucleotide corresponding to aninternal region of pir and a second oligonucleotide corresponding to aregion, close to chromosomal uidA, but not within the fragment whichserved for the recombination. The sequence of the latter oligonucleotidewas determined by means of the ECOUIDAA sequence from Genbank (accessnumber: M14641). We were thus able to verify the exact location of thepir gene in the bacterial genome. The nature of the amplified fragments,whose size is in accordance with that which might be expected, wasconfirmed by digestion with MluI.

EXAMPLE 2 Construction of Plasmid Vectors Derived from R6K Carrying theSelection Marker sup Phe

Vectors were constructed containing ori γ from R6K and thekanamycin-resistance gene (pXL2666). The observation of pXL2666multimers in the strain BW19610 (pir-116) 5 (Metcalf et al., 1993) ledus to study the effect of the cer fragment from ColE1 on thisphenomenon. We then introduced the expression cassette of thephenylalanine suppressor tRNA (sup Phe) onto the vector ori γ-Km^(R)-cer(pXL2730). This vector, pXL2760, serves as a basis for the constructionof vectors which can be used in gene therapy.

-   -   1) Construction and Analysis of Vectors Containing ori γ from        R6K and the Kanamycin-Resistance Gene        -   a) Constructs

In the first plasmid constructed, pXL2666, the kanamycin-resistance geneoriginates from pUC4K (Vieira and Messing; 1982) and the origin ofreplication, contained in a 417 bp EcoRI-BamHI fragment, originates fromthe suicide vector pUT-T7pol (Herrero et al., 1990) (FIG. 4). Thetransfer of pXL2666 into the strains BW19094 and 19610 (Metcalf et al.,1994) made it possible to show that the amount of plasmid is indeedincreased in apir-116 strain, when compared with the same plasmid in apir strain. However, the electrophoretic analysis of the undigestedplasmids shows that this increase goes hand in hand with the appearanceof a few multimeric forms. This phenomenon is quite probably associatedwith intermolecular recombination between the multiple copies of theplasmid. Thus, we constructed pXL2730 by cloning the cer fragment of thenatural E. coli plasmid, ColE1, which had been shown to permit, in cis,the resolution of plasmid dimers (Summers and Sherrat, 1984), intopXL2666. The fragment used corresponds to a 382 bp HPAII fragment fromColE1 (Leung et al., 1985). It contains a specific intermolecularrecombination site; in order to function, it involves only host proteinsincluding the recombinases XerC and XerD and the accessory factors ArgRand PepA (Stirling et al., 1988, 1989; Colloms et al., 1990). In orderto ensure that the effects observed are indeed due to the cer fragment,we also constructed the control plasmid pXL2754 in which the cerfragment has a 165 bp deletion. This deletion was shown to abolish theaction of cer on the resolution of the multimers (Leung et al., 1985).The various cloning steps leading to the construction of these plasmidsare presented in FIG. 4.

-   -   -   b) Quantitative and Qualitative Analysis of the Plasmid            Species            -   (i) Analysis by Agarose Gel Electrophoresis

Electrophoretic analysis of the different plasmids constructed allowedthe demonstration of various plasmid species, which are variableaccording to the strains used. The size of the undigested plasmids wasevaluated relative to the supercoiled DNA marker. In the pir strain(BW19094), the plasmids pXL2666, 2754 and 2730 are almost entirely inmonomeric form. The bands above each main band correspond to variousslightly less supercoiled topoisomers, as confirmed by the profileobserved after the action of DNA gyrase on pXL2730.

In the case of the pir-116 strain (BW19610), the profiles are different:with the plasmids pXL2666 and 2754 different species are observedranging from the monomer to multimers (2, 3 or 4 units), the major formbeing the dimer. After digestion with EcoRI, only the linear plasmid DNAis found; these plasmid species correspond either to plasmid multimersor to various topoisomers. However, since the size of the formsdetermined according to the supercoiled DNA marker is a whole product ofthat of the monomer plasmid, it is highly probable that they aremultimers. The formation of multimers is most probably attributable tothe pir-116 mutation, although the two strains BW19094 and BW19610 arenot strictly isogenic (BW 19610 is recA). The profile obtained withpXL2730 is different: although multimeric forms are still visible, themajor form is the monomeric form. The cer fragment can thus facilitateresolution of the plasmid multimers which we have constructed,independently of recA, in BW19610.

-   -   -   -   (ii) Analysis After Treatment with DNA Topoisomerases

In order to disprove the theory that the forms observed in the strainscarrying the pir-116 allele are specific topoisomers, each plasmidpreparation was subjected to the action of DNA topoisomerases. Theactivities of the various enzymes under the experimental conditions areas follows: relaxing of DNA for E. coli DNA topoisomerase I, negativesupercoiling of relaxed DNA for E. coli DNA gyrase, and disentanglementof interlaced DNAs and relaxation of supercoiled DNA by S. aureus DNAtopoisomerase IV. The action of DNA topoisomerase IV made it possible toshow that the high-molecular-weight plasmid forms did not result fromthe entanglement of several plasmid molecules; in this case, they wouldthen have been converted into the monomeric species. The functionalityof the enzyme was, of course, checked on a preparation of kinetoplastDNA, composed of entangled DNA molecules (not shown). The relaxationactivity is also visible since species are obtained which migrate lessthan in the untreated controls. The action of DNA gyrase made itpossible to convert the slightly relaxed topoisomers into the moresupercoiled species extracted from the bacterium (monomer or dimermainly). Furthermore, it made it possible to verify that the DNAsprepared are mainly in supercoiled form. The samples thus treated allowthe above results to be confirmed as regards the major species for eachconstruct. DNA topoisomerase I did indeed relax DNA, but only partially.This could be due to the fact that the plasmids studied contain only afew single-stranded regions, to which this enzyme preferably binds(Roca, 1995).

-   -   2) Introduction of the Selection Marker sup Phe into pXL2730

We used the expression cassette of the synthetic suppressor tRNA gene(Phe) (Kleina et al., 1990). This introduces a phenylalanine into thegrowing polypeptide chain in response to a TAG codon. Furthermore, itallows the production in XAC-1 of an ArgE protein which is sufficientlyactive to allow good growth in arginine-deficient medium. sup Phe isexpressed constitutively on the plasmid pCT-2-F (Normanly et al., 1986),from a synthetic promoter derived from the promoter sequence, Plpp, ofthe E. coli lpp gene. Downstream of this gene, transcription is stoppedby the synthetic terminator, T_(rrnC), of the E. coli operon rrnC(Normanly et al., 1986). The various cloning steps are indicated in FIG.5.

The various subclonings were performed in XAC-1. The functionality ofthe suppressor tRNA expression cassette is thus checked by means of theα-galactosidase activity of this strain, which only exists if there issuppression of the amber codon of the gene lacZ_(u118am). The final stepconsists of the introduction of the sup Phe expression cassette intopXL2730. The results obtained with the cer fragment (B-1-b) led us toselect this plasmid rather than pXL2666. We retained thekanamycin-resistance gene for ease of subsequent cloning, in particularin order to have available additional screening during the final cloning(loss of Km^(R)).\

EXAMPLE 3 Validation of the Plasmid Vector for Applications in GeneTherapy by Transfection of Mouse Fibroblasts

-   -   1) Construction of the Reporter Vector pXL2774

In order to test the validity for gene therapy of the system forproducing plasmid DNA, we introduced a reporter gene, which can be usedin eukaryotic cells, into pXL2760. We used the gene luc coding forPhotinus pyralis luciferase since the bioluminescence measurement testis very sensitive and is linear over a large range, and the backgroundnoise due to the endogenous activity of eukaryotic cells is very low.The luc gene is controlled by promoter-enhancer sequences of a humancytomegalovirus early gene (CMV promoter) which allows a high level ofexpression. There is an untranslated region at the 3′ end of luc,originating from the virus SV40, which contains the polyadenylationsignal (poly(A)+). After intermediate cloning which allows the number ofavailable restriction sites to be increased, the “CMVpromoter-luc-poly(A)+” cassette is introduced into the minimal vectorori γ-cer-sup Phe (pXL2760) in place of the Km^(R) marker. The resultingplasmid has been named pXL2774. FIG. 5 collates the various cloningsteps. The ligation mixtures were transformed into XAC-1pir-116 byelectroporation. Incubation allowing the bacteria to express selectionmarkers is carried out in rich medium (SOC medium); it was thusnecessary to wash the cells twice with M9 medium before plating out.This made it possible to remove the residual medium which would haveresulted in culture background noise on minimal medium.

The medium chosen to plate out the electroporated cells is M9 minimalmedium, which makes it possible to select bacteria expressing asuppressor tRNA and thus the presence of our plasmids. The addition ofX-Gal makes it possible, by means of the blue colouration, to visualizethe expression of the suppressor tRNA. The dishes are analysed afterabout 20 hours at 37° C. The absence of colonies on the DNA-free controlassures us that the selection is correct, even with dense seedings. Allthe clones examined by restriction (8) do indeed carry a plasmid,corresponding to the expected profile. The plasmid thus constructed,pXL2774, was prepared from a clone cultured in one litre of liquid M9medium (about 18 hours at 37° C.), by a technique involving, inter alia,an ion-exchange (Promega kit, MegaPreps). The amount of DNA collected is2 mg.

-   -   2) Analysis of the Reporter Vector pXL2774 Transfected into        Mammalian Cells.

The capacity of pXL2774 to transfect eukaryotic cells and to allow theexpression of luciferase is evaluated by transfection into NIH 3T3 mousefibroblasts. The vector chosen as reference is the plasmid pXL2622 (thisis the plasmid pGL2 from Promega whose SV40 promoter has been replacedby the CMV promoter) which carries the same luciferase expressioncassette as pXL2774, but on a different replicon. This is a 6.2 kb ColE1derivative which carries the ampicillin-resistance gene. This plasmidserves as a control. The luciferase activities (expressed as RLU, orrelative luminescence units) are indicated in Table 3.

The best results were obtained with a “lipofectant charges/DNA charges”ratio of 6; under these conditions, pXL2622 and 2774 appear to beequivalent. TABLE 3 pXL2774 pXL2622 Coefficient RLU/μg CoefficientRLU/μg of of Charge of proteins and of variation proteins and variationratios per well Average (%) per well Average (%) 0 0.0 not 0.0 not 0.0detect- 0.0 detect- 0.0 able 0.0 able 3 9.9 10⁶ 7.6 10⁶ 22 3.3 10⁶ 2.910⁶ 13 6.2 10⁶ 2.9 10⁶ 6.6 10⁶ 2.4 10⁶ 6 1.2 10⁷ 1.5 10⁷ 19 9.4 10⁶ 1.010⁷ 7 1.5 10⁷ 9.9 10⁶ 1.9 10⁷ 1.1 10⁷ 9 9.5 10⁶ 1.0 10⁷ 26 1.1 10⁷ 6.410⁶ 13 7.5 10⁶ 8.3 10⁶ 1.4 10⁷ 8.5 10⁶

EXAMPLE 4 Verification of the Suicide Vector Nature in E. coli of thepCOR Plasmids

The non-replicative nature of the pCOR-type plasmids derived from R6Kwas verified by an electroporation experiment in JM109 E. coli(Yanisch-Perron et al., 1985) of the plasmids pUC4K (ori ColEI-KmR,(Vieira and Messing, 1982)) and pXL2730 (ori gamma from R6K-KmR, seeExample 2). The electroporator used is the Biorad Gene Pulser and theelectrocompetent JM109 cells are prepared and used according to themanufacturer's procedure (Bacterial electro-transformation and pulsecontroller instruction manual. catalog number 165-2098).

The electrotransformed cells were plated out on LB medium supplementedwith kanamycin (50 mg/l) and incubated overnight at 37° C. The resultsobtained are presented below. Results Efficacy (number of Amounttransformed Number of transformants/ Plasmid (ng) transformants ng ofplasmid) pUC4K 0.01 >>2000 >2105 pXL2730 5 0 0

These results show that there is a minimum of 5 log of differencebetween the efficacy of transformation of a ColEI derivative (pUC4K) andthat of an R6K derivative (pXL2730) in a strain which does not expressthe pir gene. In a pir+ strain such as XAC-1pir-116, theelectrotransformation efficacy of R6K-derived plasmids conventionallyreaches or exceeds the 108 transformants/μg of plasmid.

EXAMPLE 5 Production of Plasmid DNA by High-Density Culturing of the E.coli Strain XAC-1pir-116 (pXL2774): Fermentation Process

-   -   5.1. Strains:

Production in XAC-1pir-116 E. coli (Example 1) of a minimal plasmid,pXL2774; this plasmid comprises the following elements: oriR6K-cer-tRNAamsupPhe and an expression cassette of the luc reporter geneunder the control of the CMV promoter (Example 3). A high-productivityprocess for the production of plasmids of this type was developed.

-   -   5.2. Culturing Media and Conditions:        -   a) Growth Medium:

Composition of the medium defined used for the inoculum cultures (g/l):Na₂HPO₄ 6, KH₂PO₄ 3, NaCl 0.5, NH₄Cl 1, NH₄H₂PO₄ 3, glucose 5,MgSO₄.7H₂0 0.24, CaCl₂.2H₂O 0.015, thiamine HCl 0.010

Composition of the complex medium used for the fed-batch cultures (g/l):KH₂PO₄ 8, K₂HPO₄ 6.3, Na₂HPO₄ 1.7, (NH₄)₂SO₄ 0.74, NH₄Cl 0.12, yeastextract 3, glucose 2, MgSO₄.7H₂0 2.4 g/l, CaCl₂.2H₂O 0.015, thiamine0.010, solution of salts (Fe, Mn, Co, Zn, Mo, Cu, B, Al).

Composition of the medium defined for the cultures in fed-batch mediumidentical to the complex medium but the yeast extract is replaced by 2.5g/l of NH₄Cl.

-   -   -   b) Conditions of Fed-Batch Culturing:

Studies in 2-litre fermenters (Setric France) containing 1 l of mediumwere carried out in order to define the optimum conditions for growingand producing plasmid DNA. The fermenter was inoculated with 80 ml of aninoculum culture arrived at the start of the stationary phase of growth.

During the fermentation, the pH was controlled and adjustedautomatically between 6.9 and 7.0 with 10% (w/v) aqueous ammonia; thetemperature is maintained at 37° C.; the aeration was set at 75 l/h((1.1 vvm) at a pressure of 0.2 bar and the dissolved oxygen wasadjusted to (40% of air saturation by retroaction on the stirring rateand, if necessary, by enrichment with pure oxygen.

All the parameters (pH, temperature, stirring, OD, O₂ and CO₂ in theeffluent gases) were collected and calculated in line via an HP3852interface connected to a Hewlett-Packard 9000.

The base composition of the supply medium is as follows: 50% carbonsource, 0.7% magnesium sulphate, 0.02% thiamine; for the complex medium,yeast extract was added to a concentration preferably of between 5 and10%.

In order to adapt the culture conditions to 800-litre fermenters,production sequences composed of two successive inoculum cultures werecarried out, on a laboratory scale: inoculum I in an agitated conicalflask and inoculum II in a 2-litre fermenter (batch culturing), followedby fed-batch production culturing, in a 7-litre fermenter.

-   -   5.3. Results

Various culture conditions were studied in complex medium, in definedmedium, and at various growth rates. In all cases, after initial batchculturing of the bacterial strain and consumption of the carbon source,the supply medium was added to the fermenter by means of a peristalticpump coupled to a pre-programmed addition profile. This profile wasdeduced from previous experiments in which the supply rate had beencontrolled either by the level of dissolved oxygen or by a constantgrowth rate.

Furthermore, in order to extrapolate without difficulty the 2-litrefermentation condition to an 8001 fermenter without overoxygenation ofthe medium, the maximum oxygen demand at the end of the culturing wasset at 2.5-3 mM/min. For this, the growth rate of the microorganism wasreduced, if necessary, by varying the supply rate of the complementarycharge.

As seen in Table 4, very good results were obtained both in complexmedium and in defined medium, both on the laboratory scale and on the800-litre fermenter scale; furthermore, the plasmid DNA growth andproduction kinetics are entirely comparable (cf. FIGS. 6 and 7). TABLE 4Defined Complex medium medium 2 or 7 l 800 l 2 l fermenter fermenterfermenter Duration of 40 39 48 fermentation (hours) μh-l 0.130 0.1320.124 OD (600 nm) 114 100 94 X g/l 44 37 30 Plasmid DNA 115 100 100(mg/l medium) Plasmid DNA 2.6 2.7 3.3 (mg/gX)X = Biomass (weight of dry cells)

From the overall results it emerges that:

-   -   changing the scale of the fermentor from 2 litres to 800 litres        can be carried out without any problem,    -   the oxygen consumed is strongly correlated to the biomass        produced (1.08 g O₂/g of biomass produced),    -   the plasmid is stable for at least 50 generations without        selection pressure,    -   a high biomass, greater than 40 g of dry cells/litre, can be        obtained in complex medium,    -   the plasmid DNA production reaches 100 mg of supercoiled DNA/l        of medium,    -   there is very good correlation between the DNA production and        the biomass: the production can be estimated to (1 mg of plasmid        DNA/OD unit, or alternatively (2.7 mg of plasmid DNA/g of        biomass, irrespective of the duration of fermentation,    -   the use of a defined medium also makes it possible to achieve a        high biomass (30 g of dry cells/l) and high plasmid DNA        production (100 mg/l), without any loss of productivity.

EXAMPLE 6 Transfer of pXL2774 into Animal Cells, in vitro and in vivo

-   -   6.1. in vitro Transfer of pXL2774 into Animal Cells

The capacity of the minimal plasmid pXL2774 to transfect various celllines was tested in vitro, on cells of both human origin and murineorigin. The pXL2784 plasmid was used as control. It contains the sameeukaryotic expression cassette (CMV promoter-luciferase-polyA) aspXL2774, but this is a 6.4 kb ColE1 derivative which comprises the genefor imparting kanamycin resistance in E. coli.

The cells tested are the following: Atcc ref./ Cells Type literatureref. 3LL Mouse pulmonary carcinoma NIH 3T3 Mouse embryo fibroblastsCCL92 293 Human embryo renal cells transformed CRL1573 with type-5adenovirus HeLa Human carcinoma from the neck of the CCL2 womb Caco-2Human colon adenocarcinoma HTB37 H460 Human lung carcinoma with no smallHTB177 cells ECV 304 Human umbilical cord endothelial cells Takahashi etal., 1990

The transfection conditions were as follows:

D-1: Inoculation of the cells at a density of 100,000 cells per 2 cm²well (24-well plate) in DMEM medium (Dulbecco's modified Eagle Medium)supplemented with 10% foetal calf serum (FCS).

D-3: Transfection of the cells, by 10 μl of a transfection solutioncontaining: 0.5 μg of DNA, 150 mM NaCl, 5% glucose and 3 nmol of RPR120535 lipofectant per μg of DNA, in 250 μl of culture medium, which is oris not supplemented with 10% FCS. After incubation for 2 hours, themedium is replaced by 500 μl of DMEM medium supplemented with 10% FCS.

D-4: Renewal of the culture medium

D-5: Washing of the cells with PBS, followed by lysis with 100 μl ofPromega lysis buffer (Promega Cell Lysis Buffer E153 A). Assay of theluciferase activity is carried out in a Lumat LB 9501 luminometer(Berthold) on 10 μl of lysate, with a 10-second duration of integration.The reactant used is that from Promega (Promega Luciferase AssaySubstrate). The results, collated in the following tables 5-8, areexpressed in RLU (Relative Lights Units) for 10 μl of lysate (average ofmeasurement on 4 wells). The coefficients of variation (CV) are alsogiven.

The results of transfections in the absence of serum are presentedbelow. CELL TYPES NIH 3T3 3LL 293 pXL2774 37 763 380 559 270 1 884 200RLU      16    25      73 CV pXL2784 113 764 1 723 546 RLU    24     101CV CELL TYPES HeLa CaCo2 H460 ECV304 pXL2774 11 000 000 1 108 422 1 459501  36 450      15      14      5     23 pXL2784   557 930   93 610   7 563 168 795      87      40      11    40

The results of transfections in the presence of serum (10%) arepresented below: CELL TYPES NIH 3T3 3LL 293 pXL2774 50 612 590 566 377  992 500      12    18       59 PXL2784 12 693 780 436 704 2 300 000     38    12      47 HeLa H460 ECV304 pXL2774 9 490 000 857 385 18 021     25    16    30 PXL2784 1 508 480 433 023 32 074      23    27    47

These results reveal the capacity of pXL2774 to transfect effectively,in vitro, various cell types of both murine and human origin. Theexpression of the luc reporter gene makes it possible to show that itstransfection efficacy is at least as good as that of a “standard”plasmid, derived from ColE1, which carries the same expression cassetteof luciferase.

-   -   6.2. in vivo Transfer, in Animals (Mice), of pXL2774        -   a) Model 1: Naked DNA in Mouse Cranial Tibial Muscle

Naked plasmid DNA, dissolved in “5% glucose, 150 mM NaCl” is injectedinto the cranial tibial muscle of OF1 mice. The muscles are removed 7days after injection, chopped up, homogenized in 750 μl of lysis buffer(Promega Cell Lysis Buffer E153A) and then centrifuged at 20,000×g for10 minutes.

Assay of the luciferase activity is carried out on 10 μl of supernatantafter addition of 50 μl of reagent (Promega Luciferase Assay Substrate).The reading is carried out on a Lumat LB9501 luminometer (Berthold) witha 10-second duration of integration.

The results are presented in the table below. Plasmid pXL2784 pXL2774pXL2784 pXL2774 Number of 8 8 10 10 muscles: Volume 30 30 33 33 injected(ìl): μg of 19 13.5 10 6.25 DNA/muscle RLU (for 10 μl) Average  80 922471 733 35329 30569 Standard 104 573 402 602 37041 35774 deviation

These results show that a conditional replication plasmid such aspXL2774 is indeed capable of transfecting mouse muscle cells in vivo andof doing so with comparable, or even superior, efficacy to that of a“standard” plasmid, derived from ColE1, which carries the sameexpression cassette of the luciferase gene.

-   -   -   b) Model 2: 3T3 HER2 Tumour Model

The model is as follows:

-   -   Swiss/nude adult female type mice    -   Experimental tumours induced after injection of 107 3T3 HER2        cells subcutaneously into the flank.    -   The transfection mixture is injected 7 days after injection of        the cells.

Solutions injected: The DNA is first dissolved in the buffer. Afteraddition of all the products, the mixture contains, besides the DNA,NaCl (150 mM) and 5% D-glucose in water or 5 mM HEPES buffer.

-   -   Two days after the injection, the tumour tissue is removed,        weighed and then chopped up and homogenized in 750 ìl of lysis        buffer (Promega Cell Lysis Buffer E153 A). After centrifugation        (20,000×g for 10 minutes), 10 μl of supernatant are removed and        allow the luciferase activity to be evaluated. This activity is        determined by measuring the total light emission obtained after        mixing with 50 μl of reagent (Promega Luciferase Assay        Substrate) in a Lumat LB 9501 luminometer (Berthold) with a        10-second duration of integration.

The resulting activity is expressed in RLU (Relative Light Units)estimated in the entire tumour lysis supernatant. Results Plasmid Buffer[DNA] RLU/tumour results H20 or final in standard HEPES referenceμg/tumour inj. sol. average deviation +/n HEPES pXL2784 10 0.5 μg/μl  744 150   682 434 6/6 pXL2774 10 0.5 μg/μl 1 016 380 1 322 500 5/6 H2OpXL2784 24 0.6 μg/μl 2 906 073 1 745 857 8/8 pXL2774 16.8 0.4 μg/μl 4292 043 4 995 187 6/6 H2O pXL2784 7.5 0.3 μg/μl   702 554   552 207 6/7pXL2774 5 0.2 μg/μl 3 413 430 4 000 875 6/6

These results show that a conditional replication plasmid, such aspXL2774, is indeed capable of transfecting mouse tumour cells in vivoand of doing so with an efficacy at least comparable to that of a“standard” plasmid, derived from ColE1, which carries the sameexpression cassette of the luciferase gene.

These various experiments made it possible to demonstrate that theconditional replication plasmids, and more particularly pXL2774, didindeed have animal cell transfection characteristics that are essentialfor use in gene therapy. More precisely, the following were shown:

1) the capacity of pXL2774 to transfect efficiently, in vitro, variouscell types, of human or murine origin;

2) the capacity of pXL2774 to transfect, in vivo, mouse muscle;

3) the capacity of pXL2774 to transfect, in vivo, tumour cells implantedinto mice.

The electrotransformation, fermentation and transfection experimentsthus made it possible to validate conditional replication plasmids asvectors which can be used in gene therapy by showing

i) that they did not replicate detectably in an E. coli strain whichdoes not express the pir gene (conditional origin of replication)

ii) that they could be produced on a scale compatible with industrialproduction, in a medium which can be totally defined and which does notcontain antibiotics;

iii) that these plasmids could transfect, in vitro and especially invivo, mammalian cells.

EXAMPLE 7 In vitro Production of Recombinant Proteins

-   -   7.1. Construction of the Expression Vector

In order to show the feasibility of such an approach, we constructed anexpression vector according to the criteria described above (Examples 2and 3). This vector, pXL3056, contains:

1) the bacterial part which comprises the conditional origin ofreplication (ori gamma), the cer fragment of ColE1 and the gene whichensures selection in bacteria (sup)

2) the expression cassette, based on the system described by Studier(Studier et al., 1990), comprises the promoter of gene 10 ofbacteriophage T7, the lacO operator, the gene coding for aFGF 154(acidic Fibroblast Growth factor, form containing 154 amino acids) (Jayeet al., 1986), the TF terminator of bacteriophage T7. This expressioncassette is identical to the one present on the pXL2434 plasmiddescribed in application WO 96/08572.

The construction of pXL3056 is presented in FIG. 8. The EcoRI-BglIIfragment of pXL2434 (1.1 kb) containing the aFGF expression cassette iscloned in the pXL2979 conditional replication vector (1.1 kb purifiedfragment) at the BglII and EcoRI sites in order to generate pXL3056.

pXL2979 results from the ligation of 3 fragments: i) AccI-XbaI fragmentof pXL2730 (0.8 kb, which provides ori gamma and cer), ii) NarI-SalIfragment of pXL2755 (0.18 kb, which provides the sup Phe gene), iii)SalI-SpeI fragment of pXL2660 (1.5 kb, which provides thekanamycin-resistance gene).

pXL2660 results from the cloning of the 1.2 kb PstI fragment of pUC4K(Vieira and Messing, 1982) in pMTL22 (Chambers et al., 1988) linearizedwith PstI.

-   -   7.2. Production of the Expression Strain

The plasmid pXL3056 is introduced by transformation into theXAC-1pir-116 strain. The resulting strain is then transformed by theplasmid PT7pol23 (Mertens et al., 1995), at 30° C. In order to expressthe gene of interest under control of the T7 promoter, the bacteriummust contain, in its genome, on a plasmid or a bacteriophage, a cassetteto allow the expression of the RNA polymerase of bacteriophage T7. Inthe example described, we used the plasmid PT7pol23, which is compatiblewith R6K derivatives such as pXL3056, and which allows thetemperature-inducible expression of bacteriophage T7 RNA polymerase.However, it can also be envisaged to lysogenize the XAC-1pir-116 strainwith lambda DE3 (Studier et al., 1990) in order to conserve only oneplasmid and to induce the production of T7 RNA polymerase by IPTG ratherthan by temperature.

-   -   7.3. Expression of AGF

The XAC-1pir-116 strain (pXL3056+PT7pol23) is cultured at 30° C., in M9minimum medium supplemented with 0.2% of casamino acids (DIFCO) andkanamycin (25 μg/ml), up to an optical density at 600 nm of 0.6-1. Halfof the culture is then placed at 42° C. (induction of the T7 RNApolymerase), while the other half remains at 30° C. (negative control).The same experiment is carried out with the XAC-1pir-116 (pXL3056+pUC4K)strain which constitutes a control for the expression of aFGF in theabsence of T7 RNA polymerase.

The results obtained are presented in FIG. 9. They show that theproduction of aFGF is comparable or superior to that observed withBL21(DE3)(pXL2434) (WO 96/08572), which clearly shows the potential ofconditional replication plasmids for the expression of recombinantproteins in vitro, especially in E. coli.

EXAMPLE 8 Construction of a pCOR Vector which Expresses a Wild-Type orHybrid p53 Protein

This example describes the construction of conditional replicationvectors according to the invention containing a nucleic acid which codesfor a p53 protein. These vectors can be used to restore a p53-typeactivity in deficient (mutated, deleted) cells such as, in particular,tumour cells.

The eukaryotic expression cassette contains the following elements:

1) CMV “immediate early” promoter (positions −522 to +72) followed bythe leader sequence of the thymidine kinase gene of type I herpessimplex virus (position −60 to +1 of the gene, with reference to thesequence in the article by McKnight, S.†L. (1980) Nucleic Acids Res.8:5949-5964);

2) a nucleic acid which codes for wild-type p53 protein or for a p53variant, as described in application PCT/FR 96/01111 (V325K variant=V325with a Kozak sequence with ATG);

3) the polyA polyadenylation sequence of SV40.

These elements were placed in the form of a fragment AscI-XbaI on thepCOR vector pXL2988 between the sites BssHII and SpeI. pXL2988 isidentical to pXL2979 (Example 7.1.) apart from the presence of anadditional element, a sequence capable of forming a DNA triple helixcomposed of 17 times the trinucleotide GAA, placed alongside the gammaorigin of replication.

The resulting plasmids are named pXL3029 and 3030 (FIG. 10).

The functionality of these constructions was verified in vitro onp53-SAOS2 cells in culture by measuring the transcriptional-activatoractivity of p53 or p53superWT.

EXAMPLE 9 Construction of TEX 1 (XAC1 pir116, endA⁻, traD-⁻)

The E. coli XAC-1pir116 contains an F′ episome, a circular DNA moleculeof approximately 100 kb, that carries proB⁺ lacI₃₇₃lacZ_(u118am). Manymale E. coli laboratory strains carry a traD36 mutation on theirepisome, but no mutation affecting F′ transfer ability has beendescribed for XAC-1. traD is at the 5′ end of one of the tra (transfer)operons and encodes a membrane protein that is directly involved in DNAtransfer and DNA metabolism (Frost et al., BBRC, 1994, 58:162-210). A 2kb central fragment from traD, comprising 92% of the gene, was replacedwith the 2 kb omega element (Genbank accession number M60473) frompHP45Ω (Prentki and Krisch, 1984, Gene, 29:303-313) by homologousrecombination in XAC-1 pir 116 endA⁻. The omega element contains theaadA antibiotic resistance gene flanked by short inverted repeats. aadAencodes aminoglycoside-3 adenyltransferase and confers resistance tostreptomycin and spectinomycin (Sp^(R)). The omega fragment was usedbecause it prematurely terminates RNA and protein synthesis leading tothe inactivation of the whole traD operon. This new pCOR strain XAC-1pir-116 endA- traD::SpR was designated TEX1. Transfer of any residentplasmids, either pCOR or pUC was undetectable when the donor was TEX1.

The new pCOR host strain TEX1 was assessed in fermentation experiments.Complex media containing yeast extract were used for fed-batchfermentation with XAC-1pir 116. pCOR stability (more than 50generations) makes it possible to use a non-selective media. Under theseconditions, XAC-1pir116 produced more than 40 g/l dry cell weight and100 mg/l of pCOR pXL2774 were obtained from 2-liter fermentors. pCORcopy number was estimated at 400-500 copies per cell and the rate ofplasmid DNA synthesis was constant throughout fermentation. Theseresults were extrapolated to an 800-liter fermentor suitable formanufacturing. The fermentation was also performed in the absence ofyeast extract or any raw material from animal origin. Similar results(30 g/l dry cell weight and 100 mg/l of plasmid DNA) were obtained usinga defined medium in 2-liter cultures with no loss of productivity.

EXAMPLE 10 Construction of XAC-1pir-116 pir42 Host Strains by HomologousRecombination

-   -   1) Construction of a Suicide Vector Carrying the Cassette        “KmR-uidA:pir116;pir-42”

The Km^(R)-uidA::pir-116 cassette from M13wm33 as described in Example 1(Metcalf W. et al. Gene, 1994, 138(1-2): p. 1-7), was modified bysite-directed mutagenesis using PCR (QuickChange site-directedmutagenesis kit, Stratagene, La Jolla, Calif.) in order to introduce thepir42 mutation into the pir116 gene. The different cloning/mutagenesissteps are described in FIG. 13

The oligonucleotides used for mutagenesis contained the pir42 mutationalong with a silent mutation that created a ClaI site to easily indicatethe processing of pir42 by restriction analysis when needed.

The sense and antisense oligonucleotides used are as follows: Senseoligonucleotide number 11076 9 5′-G TAT ATG GCG CTT GCT CTC ATC GAT (SEQID NO: 7) AGC AAA GAA CC-3′          pir42 ClaI Antisenseoligonucleotide number 11077 5′-GG TTC TTT GCT ATC GAT GAG AGC (SEQ IDNO: 8) AAG CGC CAT ATA C-3′                  ClaI pir42

The technique used to replace 116 by pir116-pir42 in the genome of E.coli pCOR host TEX1 was based on that of Blum et al. (J. Bacteriol.1989, 171, pp 538-46). The recombinant bacteriophage pXL3723 shown inFIG. 13 is a suicide vector in all non-suppressor E. coli strains,because it has a non-sense mutation in gene II encoding M13 nickase thatprevents viral genome replication.

Double recombination was performed as described for the construction ofXAC-1pir116 (Example 1, point 2). Clones that had undergone doublehomologous recombination events were screened by PCR to test for thepresence of the pir42 mutation in the genome of TEX1. Genomic DNAisolated from double recombination candidates was used as a template forPCR. Secondly, sequencing was done on each unique amplified fragment,all of which were of the expected size. The PCR fragments are shown inFIG. 14.

The PCR primers were the following: Primer 11088 (SEQ ID NO 9):5′-GAGATCGCTGATGGTATCGG-3′ Primer 11089 (SEQ ID NO:10):5′-TCTACACCACGCCGAACACC-3′

This analysis showed that one out of the six double recombinants testedhad undergone the allele exchange. This new strain was named TEX1pir42and further evaluated for its ability to replicate or not pCOR plasmidscompared to the parental strain TEX1.

-   -   2) Evaluation of TEX1pir42

pCOR plasmids were transformed in parallel into TEX1 and TEX1pir42 andgrown overnight in 2 ml of selective M9 medium. Then, the plasmid DNAwas extracted with the Wizard SV plus minipreps kit (Promega) in orderto evaluate the relative plasmid copy number and topology of the pCORplasmids in both strains.

A 2-fold increase in copy number was obtained reproducibly in TEX1pir42transformed with the pCOR plasmid pXL3516 (2.56 kb). To furthercharacterize TEX1p42, the copy number and topology of pCOR plasmids suchas pXL3179 and pXL2774 were evaluated by agarose gel electrophoresisanalysis after small scale purification of plasmid DNA (4 to 6clones/strain). Copy number was evaluated on plasmids linearized withEcoRI restriction enzyme. A topology test was run on non-digestedplasmids, in the absence of ethidium bromide. The resulting agarose gelis displayed in FIG. 15, and clearly shows a higher plasmid copy numberwhen the plasmid pXL3179 was produced in TEX1pir42, than when producedin TEX1 strain. FIG. 15 also displays the topology of the plasmidpXL3179, and shows that an increase in plasmid copy number, which areessentially in the form of monomers, with few plasmids in the form ofmultimers. The results obtained with these pCOR plasmids are alsosummarized in Table 5. Relative copy number was calculated in comparisonwith the same plasmid in TEX1. A 2-3 fold increase in plasmid copynumber was observed with plasmids pXL3179 and 2774 produced inTEX1pir42. TABLE 5 Replication and copy number of pCOR plasmids producedin TEX1pir42 RELATIVE COPY PLASMIDS SIZE (kb) NUMBER* pXL3179 2.4 x3pXL2774 4.5 x2*copy number was compared to the same plasmid in TEX1.

EXAMPLE 11 Comparative Experiments: Construction of TEX1cop21(XAC-1endA−traD− pir116cop21)

-   -   1) Construction of TEX1cop21

The TEX1cop21 strain was constructed similarly as that described inExample 10 for TEX1pir42. The following oligonucleotides used tointroduced 21 into the pir116 gene by directed mutagenesis were asfollows: Sense oligonucleotides: 11153 5′-CG CAA TTG TTA ACG TCC AGC TTA(SEQ ID NO: 11) CGC TTA AGT AGC C-3′              cop21 Antisenseoligonucleotide: 11154 5′-G GCT ACT TAA GCG TAA GCT GGA (SEQ ID NO: 12)CGT TAA CAA TTG CG-3′

The cop21 mutation was introduced as a TCC Serine codon instead of theTCA Serine codon to eliminate a HindIII restriction site close to themutation.

The template used for directed mutagenesis was pXL3395 (See FIG. 13).The resultant plasmid named pXL3432 was used to construct the suicideM13 vector in a similar way as what is described for pir42 in FIG. 13.The suicide vector pXL3749 is presented in FIG. 16.

The E. coli clones obtained after homologous recombination with pXL3749were screened by PCR and subsequent restriction with HindIII andsequencing to monitor the cop21 and pir116 mutations. One clone out ofthe six double recombinants tested had undergone the gene replacement.The resulting strain was named TEX1cop21.

-   -   2) Evaluation of TEX1cop21.

TEX1op21 was transformed by various pCOR plasmids, including pXL2979, a2.5 kb Km^(R) pCOR (See Example 7.1), and was assayed for increased copynumber by gel electrophoresis. Such an experiment with pXL2979 is shownin FIG. 17. Plasmid DNA from four independent clones for each strainprepared with Promega miniprep kit was linearized with EcoRI restrictionenzyme, electrophoresed on agarose gel and then stained with ethidiumbromide. Each sample represented a similar amount of bacteria, asmeasured by optical density at 600 nm. The agarose gel electrophoresisobtained for the pCOR plasmid pXL2979 produced in E. coli TEX1cop21,XAC1pir, and TEX1 is displayed in FIG. 17. It clearly shows there was noincrease in plasmid copy number when the plasmids are produced in theTEX1cop21 strain, as compared with TEX1.

EXAMPLE 12 Construction of TEX2pir42 (XAC-1 pir116 pir42 recA⁻)

Firstly, a recA− derivative of TEX1 was constructed. The pir42 mutationwas then introduced into the resulting strain named TEX2 to generateTEX2pir42.

-   -   1) Construction of E. coli TEX2, a recA− Derivative of TEX 1

A deleted recA gene containing 3 translation stop codons (one in eachframe) at its 5′ end was obtained by PCR. This deleted recA gene wasintroduced by gene replacement (Blum et al., J. Bacteriol., 1989, 171,pp. 538-46) into the TEX1 genome. The construction of the suicide vectorfor homologous recombination is shown in FIG. 18.

PCR primers used for the amplification of recA fragments were thefollowing Table 6: TABLE 6 primers DNA sequences seq 109305′CCCTCTAGATCGATAGCCATTTTTACTCCTG 3′ SEQ ID NO: 13 seq 109315′CGGGATCCTGATTATGCCGTGTCTATTAG 3′ SEQ ID NO: 14 seq 109325′CCCAAGCTTCTTCGTTAGTTTCTGCTACGCCTTCG SEQ ID NO: 15 C 3′ seq 109335′GGTCTAGAACGTGAAAGTGGTGAAGAACAAAATCG SEQ ID NO: 16 3′

Restriction sites added to the recA sequence are underlined.

In order to maintain the RecA+ phenotype necessary for homologousrecombination to occur, the recA function was provided to E. coli TEX1with a plasmid containing a heterologous recA gene that can complementE. coli recA mutants, such as for example the recA gene of the bacteriumAgrobacterium radiobacter, and an antibiotic gene resistance, such asthe ampicillin resistance gene. After gene replacement, the plasmid waseliminated from the recombinant strain by culture-dilution innon-selective medium (LB). The absence of the plasmid was screened forthe loss of antibiotic resistance.

The resulting strain was named TEX2. Gene replacement was monitored byPCR in FIG. 19. PCR Primers are described in the following Table 7.TABLE 7 Primers 11355-11354 Primers 11355-11354 Wild type recA 1117 bp1089 bp Deleted recA  404 bp  376 bp

The first primer was based on the sequence of the recA gene. The secondone was based on a sequence close to but outside the homology regionpresent in the suicide vector pXL3457 (immediately 5′ or 3′ of recA) toensure that amplification can only occur on a genomic fragment. Thesequence of both oligonucleotides was chosen according to the sequenceof E. coli which comprises the recA locus (Genbank ECAE000354).

The PCR fragments obtained from a recA-deleted strain are shorter ascompared to those obtained with a wild-type strain, as presented in thefollowing Table 8. TABLE 8 PCR primers for amplification of recA primers5′->3′ sequence seq 11352 - GCGACCCTTGTGTATCAAAC SEQ ID NO: 17 seq11353 - GGTATTACCCGGCATGACAG SEQ ID NO: 18 seq 11355 -GTGGTGGAAATGGCGATAGG SEQ ID NO: 19 seq 11354 - GCGATTTTGTTCTTCACCAC SEQID NO: 20

The PCR profile obtained was as expected and demonstrated the presenceof a truncated recA gene in the genome of TEX2. RecA- phenotype(sensitivity to UV light), as well as phenotypic characteristics of TEX2were checked. Phenotypic characteristics of TEX2 were the same as thoseof TEX1 strain, i.e., ara- , Rif^(R), Nal^(R), Sp^(R), UidA-, Arg-,Km^(S) Amp^(S)), as expected.

-   B) Construction of E. coli TEX2pir42

A TEX2pir42 strain was constructed by double homologous recombination,according to the strategy described in Example 10, with the exceptionthat recombination in TEX2 was carried out in presence of a plasmidcarrying a heterologous recA gene capable of complementing the the E.coli recA mutants, in order to maintain a RecA+ phenotype required forhomologous recombination.

Gene replacement was monitored by restriction analysis of the PCRproduct digested with ClaI (see FIG. 14). Gene replacement had occurredin two out of the four-studied double recombinant clones.

-   C) Evaluation of E. coli TEX2pir42    -   1) Evaluation at Lab Scale Plasmid Production:

TEX2pir42 was transformed by the pCOR plasmid pXL3179 (2.4 kb).Production of pXL3179 in TEX2p42 was intensively studied at the labscale, in terms of reproducibility of the improvement of plasmid copynumber, conditions of culture, as well as stability (number ofgenerations). All the studies consistently showed a 2 to 5-fold increaseof plasmid copy number as compared to production of pXL3179 in TEX1 inthe same conditions. Plasmid copy number was assessed further to theproduction of pXL3179 in TEX2pir42, and TEX1pir42 and TEX1 ascomparative experiments. In this experiment, plasmids were extractedfrom identical bacterial biomass, based on the OD at 600 nm, andanalyzed by agarose gel electrophoresis. The gel was stained withethidium bromide after electrophoresis. The agarose gel electrophoresis,which is displayed in FIG. 20, clearly shows that plasmids are producedin TEX2pir42 at high copy number, and advantageously shows that plasmidmultimers are reduced when produced in TEX2pir42 instead of TEX1pir42.

-   -   2) Evaluation in Fermentors:

These results were confirmed at a larger scale in 7-liter fermentors, asdescribed below.

-   -   -   a) Composition of Fermentation Media

The composition of the medium used for inoculum cultures was: Na₂HPO₄ 6g/l, KH₂PO₄ 3 g/l, NaCl 0.5 g/l, NH₄Cl 1 g/l, NH₄H₂PO₄ 3 g/l, glucose 5g/l, MgSO₄,7H₂0 0.24 g/l, CaCl₂,2H₂0 0.015 g/l, thiamine HCl 0.010 g/l.

The composition of the medium used for fed-batch culture was as follows:KH₂PO₄ 8 g/l, K₂HPO₄ 6.3 g/l, Na₂HPO₄ 1.7 g/l, NH₄Cl 2.5 g/l, glucose 10g/l, MgSO₄,H₂0 2.6 g/l, thiamine 0.011 g/l, Biospumex36 antifoam 0.1ml/l, salt mix (see table 9) 2.5 ml/l. TABLE 9 Composition of salt mixFinal concentration in Salt mix Solution fed-batch medium (g/100 ml)(mg/l) FeSO₄, 7H₂O 1.6 40 CaCl₂, 2H₂O 1.6 40 MnSO₄, H₂O 0.4 10 CoCl₂,6H₂O 0.16 4 ZnSO₄, 7H₂O 0.08 2 MoO₄Na₂, 2H₂O 0.072 1.8 CuCl₂, 2H₂O 0.041 H₃BO₃ 0.02 0.5 AlCl₃, 6H₂O 0.04 1The composition of the supply medium was as follows: 50% glucose, 0.7%,magnesium 0.02% thiamine-HCl, 1% Biospumex36 antifoam.

-   -   -   b) Fermentation Parameters

A 7-liter fermentor containing 3 liters of the fed-batch medium wasinoculated with 1.2% of the inoculum culture. Inoculum was prepared asfollows: 250 ml of the inoculum medium in a 2-liter flask was inoculatedwith 0.25 ml of a frozen cell suspension of the E. coli strain TEX2pir42(pXL3179).

Flasks were incubated for 24 hours at 37° C. at 220 rpm. After 24 hours,different parameters were measured: residual glucose: 0 g/l, OD_(600nm)was 2.7 and pH 6.24. During fermentation, the pH was controlled andadjusted automatically between 6.9 and 7 with NH₃. The temperature wasmaintained at 37° C. and the dissolved oxygen adjusted to a 45% pO2 byretroaction on the stirring rate.

After initial batch culturing of the bacterial strain for about 17 hoursand consumption of the carbon source (glucose), the supply medium wasadded. Glucose and acids such lactate and acetate were maintained at aconcentration close to 0.

-   -   -   c) Results

Final results are presented in Table 10, as compared to production in a100-liter fermentor with E. coli TEX 1 (pXL3179) in optimizedconditions.

As for XAC-1pir116, there was no difference between 7-liter and800-liter fermentors in terms of plasmid copy number of pXL3179 producedin TEX1 ( ).

Plasmids pXL3179 so produced in a 7-liter fermentor using a E. coliTEX2pir42 was compared to the production of pXL3179 in a 100-literfermentor with E. coli TEX1, in optimized conditions. It wasdemonstrated that as for the XAC-1 pir116 (See Example 5.3), there is astable plasmid production rate in a 7-liter, 100-liter, or 800-literfermentor in TEX1. TABLE 10 Characteristics of the fermentation of TEX1and TEX2pir42 strains containing pXL3179 Estimated Copy Duration ofFinal OD Cell Dry Concentration of Number Reference fermentation (h)(600 nm) Weight DNA (mg/l) (copy/bacterium) TEX1 OpGen 43.00 104 33.1 96616-627 (pXL3179) 090 TEX2pir42 Op132 48.47 72 27.1 205 1896-1904(pXL3179) 8S5

There were 3-fold more copies of plasmid pXL3179 per bacterium inTEX2pir42 as compared to TEX 1.

Plasmids corresponding to different fermentation time points wereextracted from identical bacterial biomass, based on the OD at 600 nm,and analyzed by agarose gel electrophoresis (FIG. 21). FIG. 21 clearlyshows an increase of the plasmid copy number with the duration of thefermentation. Also, FIG. 21 shows the topology of the pXL3179 plasmidproduced in Op1328S5 TEX2pir42, which was nearly exclusively in amonomeric form.

In conclusion, the E. coli host strain TEX2pir42 according to thepresent invention provided an unexpectedly high plasmid copy numberimprovement of pCOR plasmids, such as pXL3179, of 2 to 5-fold inTEX2pir42 as compared to TEX1, at a lab scale and in fermentors.Furthermore, while the plasmid copy number was greatly improved,plasmids so produced exhibited a monomeric topology, not only atlab-scale but also at a larger scale (7-liter fermentor) compatible withindustrial production.

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1-18. (canceled)
 19. A prokaryotic recombinant host cell comprising aheterologous replication initiation protein that activates a conditionalorigin of replication, wherein the heterologous replication initiationprotein is encoded by gene pir116.
 20. The prokaryotic recombinant hostcell according to claim 19, further comprising a gene comprising anamber mutation.
 21. The prokaryotic recombinant host cell according toclaim 19, wherein the host cell is XAC1pir116.
 22. The prokaryoticrecombinant host cell according to claim 19, further comprising anextrachromosomal DNA molecule, wherein the extrachromosomal DNA moleculecomprises a heterologous therapeutic gene and a conditional origin ofreplication, and wherein the conditional origin of replication isactivated by the heterologous replication initiating protein.
 23. Theprokaryotic recombinant host cell according to claim 22, wherein theheterologous therapeutic gene is under the control of a eukaryoticpromoter sequence.
 24. The prokaryotic recombinant host cell accordingto claim 22, wherein the heterologous therapeutic gene is a eukaryoticgene.
 25. The prokaryotic recombinant host cell according to claim 22,wherein the heterologous therapeutic gene encodes a viral antigenicpeptide or a tumor antigenic peptide.
 26. The prokaryotic recombinanthost cell according to claim 22, wherein the heterologous therapeuticgene is selected from a gene coding for a blood derivative, a hormone, alymphokine, a growth factor, a neurotransmitter, a trophic factor, anapolipoprotein, a tumor suppressor protein, a coagulation factor, animmunoglobulin or fragment thereof, an RNA ligand, or an antisensesequence.
 27. The prokaryotic recombinant host cell according to claim22, wherein the extrachromosomal DNA molecule further comprises aselection gene that does not impart resistance to an antibiotic.
 28. Theprokaryotic recombinant host cell according to claim 19, furthercomprising an endA gene, wherein the endA gene is inactivated.
 29. Theprokaryotic recombinant host cell according to claim 19, furthercomprising a traD gene, wherein the traD gene is inactivated.
 30. Theprokaryotic recombinant host cell according to claim 19, furthercomprising a recA gene, wherein the recA gene is inactivated.
 31. Theprokaryotic recombinant host cell according to claim 28, furthercomprising a traD gene, wherein the traD gene is inactivated.
 32. Theprokaryotic recombinant host cell according to claim 31, furthercomprising a recA gene, wherein the recA gene is inactivated.
 33. Theprokaryotic recombinant host cell according to claim 22, wherein theconditional origin of replication is from a bacterial plasmid or abacteriophage.
 34. The prokaryotic recombinant host cell according toclaim 33, wherein the conditional origin of replication is from R2K,R6K, R1, pSC101, Rts1, F, RSF1010, P1, P4, lambda, Phi82 or Phi80. 35.The prokaryotic recombinant host cell according to claim 34, wherein theconditional origin of replication is from bacterial plasmid R6K.
 36. Amethod for producing an extrachromosomal DNA molecule, comprising: a)culturing the host cell according to claim 22 under conditionspermitting selection of the host cell containing the extrachromosomalDNA molecule; and b) isolating the extrachromosomal DNA molecule.
 37. Amethod for producing an extrachromosomal DNA molecule, comprising: a)culturing the host cell according to claim 24 under conditionspermitting selection of the host cell containing the extrachromosomalDNA molecule; and b) isolating the extrachromosomal DNA molecule.
 38. Aprokaryotic recombinant host cell XAC1pir116, which was deposited at theCollection Nationale de Cultures De Micro-organisms on October 2003,under accession no. CNCM 1-3108.